Magnetic nanovectors for drug delivery

Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37 – S50

Review

nanomedjournal.com

Magnetic nanovectors for drug delivery Jim Klostergaard a,⁎, Charles E. Seeney b a

University of Texas MD Anderson Cancer Center, Houston, Texas, USA b NanoBioMagnetics, Inc., Edmond, Oklahoma, USA Received 13 December 2011; accepted 25 January 2012

Abstract Nanotechnology holds the promise of novel and more effective treatments for vexing human health issues. Among these are the use of nanoparticle platforms for site-specific delivery of therapeutics to tumors, both by passive and active mechanisms; the latter includes magnetic vectoring of magnetically responsive nanoparticles (MNP) that are functionalized to carry a drug payload that is released at the tumor. The conceptual basis, which actually dates back a number of decades, resides in physical (magnetic) enhancement, with magnetic field gradients aligned non-parallel to the direction of flow in the tumor vasculature, of existing passive mechanisms for extravasation and accumulation of MNP in the tumor interstitial fluid, followed by MNP internalization. In this review, we will assess the most recent developments and current status of this approach, considering MNP that are composed of one or more of the three elements that are ferromagnetic at physiological temperature: nickel, cobalt and iron. The effects on cellular functions in vitro, the ability to successfully vector the platform in vivo, the anti-tumor effects of such localized nano-vectors, and any associated toxicities for these MNP will be presented. The merits and shortcomings of nanomaterials made of each of the three elements will be highlighted, and a roadmap for moving this longestablished approach forward to clinical evaluation will be put forth. © 2012 Elsevier Inc. All rights reserved. Key words: Magnetic nanovectors; Magnetically responsive nanoparticles; Nanomedicine; Drug delivery; Cancer; Magnetic field gradients; Nickel; Cobalt and Iron

Targeted therapy for cancer has today evolved to primarily mean development of specific and potent inhibitors of signaling pathways that are altered or over-expressed in particular tumor cells compared to normal cell counterparts. Although there have certainly been notable gratifying successes that have thoroughly validated this approach (e.g., Herceptin, Gleevec), limitations have also been revealed – limitations that are best attributed to the plasticity and genetic instability of tumor cells. Thus, successful blockade by a specific inhibitor of an initially critical single pathway all too frequently results in emergence of tumor cells which are able to circumvent this blockade by upregulation of alternate, parallel or cross-communicating pathways. For the patient, this can be manifested in strong initial responses to treatment that are of regrettably short duration, followed by failure of continued treatment with the targeted agent.

This article is part of a supplement on “Nanotechnology: From Fundamental Concepts to Clinical Applications for Healthy Aging,” and is dually published in Maturitas. ⁎Corresponding author. E-mail address: [email protected] (J. Klostergaard). URL: http://www.nanobmi.com (C.E. Seeney).

This dilemma has led to the development of second generation therapeutics capable of inhibiting multiple pathways at once: in some sense, a slight step back from exquisite specificity toward the era of non-specific cytotoxic drugs. Conventional cytotoxic agents do not demonstrate predominant specificity for targets uniquely expressed in tumor compared to normal cells, but rely largely on favorable biodistribution to achieve their therapeutic index. One means to enhance this distribution is to use magnetically responsive nanoparticles MNP loaded with such cytotoxic drugs and directed by magnetic field gradients to the interstitial tumor microenvironment. This review will examine developments with nanoparticles based on Co, Ni or Fe, including their oxides, selected hybrids and other composites. These are the three elements that display ferromagnetic properties under physiological conditions, and the most recent developments in this field, with particular emphasis on studies that have demonstrated pre-clinical, proof-of-principle will be reviewed. The concept of magnetically vectored therapeutics has itself evolved from the use of generalized (non-focused) magnetic field gradients that deposited MNP with adsorbed therapeutics in the vicinity of the target site, relying on diffusion mechanisms for efficacy, to today's successes with localized magnetic delivery

1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nano.2012.05.010 Please cite this article as: J. Klostergaard, C.E. Seeney, Magnetic nanovectors for drug delivery. Nanomedicine: NBM 2012;8:S37-S50, http://dx.doi.org/ 10.1016/j.nano.2012.05.010

S38

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

Figure 1. Schematic representation of dynamics of passive vs. magnetically enhanced tumor extravasation of nanoparticle drug carriers. Flow in blood vessel is from bottom to top of figure panels. Arrows from vessel to clearance organs at lower left depict inverse relationship between the proportion of particles subjected to these mechanisms and the proportion available to accumulate in tumors, either passively (Panel B) or under magnetic influence (Panels C and D; magnet to the right of the vessel). Note that the blood vessel in light background of normal tissue is lined with endothelial cells that form tight gap junctions, limiting fluid and macromolecule movement; in contrast, in the tumor shaded darkly, there exist pores permeable to nanoparticles, allowing their egress into the tumor interstitial fluid (TIF).

and tumor extravasation. Magnetic vectoring of therapeutics has several contributing factors: • The capacity of shaped magnetic field gradients to focus on a target site for localized delivery at a distance which is a function of field strength and magnet configuration. • The magnetic susceptibility of the MNP component, and its capacity to be manipulated/concentrated from flow in the bloodstream, leading to extravasation. • The surface chemistry of the MNP that allows sufficient loading of therapeutics that can be deposited at the tumor site with high dose effectiveness. • The reduction/elimination of cytotoxicity effects of MNPtherapeutic prodrug constructs until within the target environment, minimizing harm to normal tissue.

Magnetic theory: 101 Magnetic theory can be quite complex, with different metal and metal oxides exhibiting varying forms of magnetism, and responding to magnetic forces in different ways. Magnetism is essentially the atomic or subatomic response of a material to an applied magnetic field, in which the electron spin and charge create a dipole moment and magnetic field. When multiple

dipoles are combined, a magnetic field of measurable intensity is created. The response to an external magnetic force can be either attraction (paramagnetism) or repulsion (diamagnetism) or something more complex. The structure of magnetic materials is based on small areas called magnetic domains, which align themselves in an applied magnetic field. With paramagnetic materials (ferromagnetic and ferromagnetic), spontaneous magnetization occurs, resulting in either the formation of permanent magnets or gradual demagnetization (remanence). However, with superparamagnetic materials, such as magnetic nanoparticles that are the size of magnetic domains, spontaneous magnetization and demagnetization occur. The responses to an applied magnetic field, while similar to paramagnets, are much larger. Superparamagnetic behavior thus gives rise to the concept of a “biomagnetic switch”, which can be exploited in the development of smart drug delivery systems. An example of the development of smart nanoparticle-based drug delivery is depicted in Figure 1. Small liposomes (Panel A) passively permeate through the pores, typically 400–800 nm in diameter, found in the tumor vasculature and accumulate in the tumor interstitial fluid (TIF), from where their drug contents may be released-or they may be internalized by tumor and stromal cells bathed by this fluid. The effectiveness of this passive mechanism is limited both by clearance of the particles from the

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

systemic circulation, such as by liver and kidney functions, and also by the high pressure of the TIF. The former limitations are somewhat mitigated by endowing the liposomes with “stealth” characteristics (Panel B), structural features that mitigate opsonization of the liposomes as a first step toward capture and engulfement by phagocytic cells. Nevertheless, this is still a passive mechanism, limited by high TIF pressure. Magnetic vectoring of MNP carriers (Panels C and D), such as those that are the focus of this review, is capable of challenging these limitations of passive targeting. In response to the gradient of the magnetic field applied orthogonally at the right of the blood vessel, the previously randomly moving particles are deflected toward the vessel wall closest to the magnet: thereby increasing the frequency and intensity of collisions with the wall and likelihood of penetrating the pores and leaving the vessel (extravasating) and reaching the TIF, despite the backpressure. Morevover, their distribution within the TIF is not limited to post-extravasation diffusion. Particles that are not so captured will continue to circulate, subject to the previously mentioned clearance mechanisms, so stealth characteristics will help increase the proportion that remain in circulation, available for additional passes through the magnetic “gauntlet”.

S39 20,26-29

mation-related signatures and cytokines, and acute oxidative stress 14 suggest significant host alterations could occur with even pharmacological exposure in the contest of cancer therapy. For examples, Ni-based nanoparticles demonstrated some characteristics of carcinogenic Ni compounds, including cytotoxicity and sustained activation of the HIF-1α pathway, associated with oncogenic transformation and tumor progression. 25 The complexity of responses to Ni-based nanoparticles was also revealed on endothelial cells (EC) following in vitro exposure. 28 Exposure led to concentrationdependent cytotoxicity, but effects on oxidative stress and proinflammatory responses diverged; further, the metallic nanoparticles and corresponding metal ions also demonstrated divergent effects, indicating differential induction of signaling. In summary, the currently limited development of Ni-based nanomaterials as magnetically responsive therapeutic platforms, when viewed in the contexts of modest magnetic susceptibility and the significant potential for host alterations that have already been revealed, suggest that this approach with nickel is not the most attractive.

Cobalt-based nanoparticles Fabrication and activity in pre-clinical tumor models in vitro

Nickel-based nanoparticles Fabrication and activity in pre-clinical tumor models Nickel has seen relatively limited assessment as a nanomaterial for platforms for tumor targeting, perhaps due to its modest magnetic susceptibility (55 emu/g of elemental Ni). Most of these studies have involved Ni oxides, 1,2 hybrid composites with Co 3 or Fe, 4-6 or with arsenic ions. 7,8 In these studies, the Nibased nanoparticles are either serving a role as inducers of hyperthermia in response to an externally applied magnetic field, 3 as drug delivery platforms 1,3,5,6,8-10 or directly as proapoptotic mediators. 6,7 Some of these approaches have matured to in vivo applications in pre-clinical tumor models, albeit only defining localization parameters in an orthotopic human glioma xenograft model 9 and a s.c. epidermoid carcinoma xenograft model 10 following systemic administration. Nevertheless, progression to an Investigative New Drug (IND) application track with any lead Ni-based formulation still appears to be somewhat in the distant future. Toxicology Despite the limited development to date of Ni-based nanoparticles as platforms for magnetically targeted treatment of tumors, the potential toxicities of such nanomaterials have received far greater attention. Many studies have focused on characterization of effects in respiratory and airway models, reflecting public health priorities regarding occupational and environmental exposures, 11-24 beyond the scope of this review. Nevertheless, effects of intracellular Ni exposure on signaling pathways, e.g., hypoxia inducible factor (HIF)-1α, 25 gene expression of matrix metalloproteinases (MMP)-2 and tissue inhibitor of metalloproteinases (TIMP)-2 mRNA 12 and inflam-

The interest in MNP based on cobalt is in part due to its high magnetic susceptibility (160 and 68 emu/g of elemental Co and Co ferrite, respectively). Co ferrite nanoparticles have been developed for the purpose of internalization into target cells, such as stem cells, intended for magnetically driven guidance to desired tissues in vivo. 30 Subsequently, 31 similarly composed nanoparticles were characterized by multiple physical–chemical techniques and magnetic measurements demonstrated the potential for hyperthermic applications following magnetic vectoring to tumors. Papis et al 32 investigated Co oxide nanoparticles and evaluated their cytotoxicity toward human cell lines. They observed a concentration- and time-dependent loss of cellular viability following their rapid uptake into cytoplasmic vesicles; however, Co ions were more cytotoxic. Conversely, these nanoparticles caused rapid reactive oxygen species (ROS) induction compared to Co ions. Block copolymers of poly(trimethylsilyl propargyl methacrylate)-block-poly(poly(ethylene glycol) methyl ether methacrylate), following removal of the TMS groups, yielded a polymer with pendant alkyne groups, which self-assembled into b20 nm micelles. 33 The core alkyne groups acted both as a ligand for Co2(CO)8, to generate a derivative of antitumor agents based on (alkyne)Co2(CO)6, and as an anchor point for the cross-linking of micelles via click chemistry. The cross-linking left defined numbers of alkyne groups that formed Co complexes. The resulting Coloaded non-cross-linked micelles were highly cytotoxic to L929 fibrosarcoma cells in vitro. Sherlock et al 34 evaluated the cytotoxicity of high magnetization CoFe/graphitic carbon shell nanocrystals, ∼5 nm in diameter. Doxorubicin (DOX) was loaded by π-stacking on the graphitic shell to afford CoFe/carbon shell– DOX complexes that demonstrated pH-sensitive DOX release. They observed enhanced intracellular DOX release following near infrared (NIR) laser-induced hyperthermia, resulting in increased

S40

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

cytotoxicity toward breast cancer cells in vitro due to both enhanced nanoparticle uptake and the increased DOX cytotoxicity under hyperthermic conditions. Liu et al 35 synthesized a Co and Cu-based nonviral carrier for DNA-transfer in gene therapy. Strong binding to DNA involved both electrostatic attractions and the intercalation of the ligands between DNA base pairs; the complexes efficiently condensed free DNA into globular nanoparticles. Cellular uptake experiments demonstrated the potential of the complexes as nonviral gene carriers, not negatively impacted by serum proteins. Wu et al 36 synthesized multi-walled carbon nanotube Co ferrite magnetic hybrids by a solvothermal method. The superparamagnetic and hydrophobic nanotubes coated with 6 nm Co ferrite nanoparticles showed high T-2 relaxivity, significant negative contrast enhancement on tumor cells and low toxicity and negligible hemolytic activity. DOX was loaded onto the hybrids, could be subsequently released in a sustained and pHresponsive manner, and exhibited cytotoxicity to HeLa cells after internalization of the hybrids. These investigators proposed that the hybrids could serve both as effective MRI contrast agents and drug delivery vehicles for simultaneous cancer theranosis. Activity in pre-clinical tumor models in vivo Scarberry et al 37 developed magnetic Co ferrite nanoparticles functionalized with ligands specific for EphA2 receptors on ovarian cancer cells; targeting resulted in cell capture from a flow stream in vitro and from the mouse peritoneum ex vivo. This approach was proposed for removal of metastatic cancer cells from the abdominal cavity and circulation via dialysis-like treatment. Pouponneau et al 38 conducted a proof-of-concept preclinical study involving the magnetic intravascular steering of micron-sized carriers designed for liver chemoembolization. The biodegradable microparticles were loaded with Co/Fe nanoparticles and DOX, and displayed high saturation magnetization (72 emu/g). They were successfully steered in vitro and in vivo, the latter involving targeting of the right or left rabbit liver lobes, illustrating the potential of magnetic resonance navigation to improve deep tissue drug targeting. Toxicology There has been far more effort in characterizing the toxicity of Co-bearing nanoparticles compared to those composed of Ni. For example, cytotoxic and genotoxic effects of nanoparticles and micron-sized particles of Co/Cr were compared using human fibroblasts. 39 Nanoparticles caused more free radicals in an acellular environment and induced more aneuploidy than micron-sized particles. Nanoparticles decomposed intracellularly more rapidly than microparticles with the creation of Coenriched electron dense deposits. Overall, the mechanisms of cell damage appear to be different for nanoparticles vs. microparticles. In an intriguing study, Co/Cr nanoparticles were found to damage human fibroblasts across an intact cellular barrier without traversing it. 40 The damage involved transmission of purine nucleotides and intercellular signaling within the barrier through connexin gap junctions or hemichannels and pannexin channels; damage occurred without cell death, distinct from that from direct exposure to nanoparticles.

Attempting to model joint replacement and to determine whether adverse biological effects occurred following exposure to particulate wear debris, Tsaousi et al 41 investigated the in vitro genotoxic effects of aluminum oxide ceramic particles and compared them to CoCr alloy particles. Primary human fibroblasts were exposed to the ceramic nanoparticles or CoCr particles; no significant differences in cell viability between control and ceramic-treated cells were observed. In contrast, fibroblasts exposed to CoCr alloy particles showed dose- and time-dependent cytotoxicity. There was only a small increase in micronucleated binucleate cells after treatment with ceramic particulates, but the increase was much greater with CoCr particles, and showed dose-dependence. A parallel pattern was observed with the numbers of γ-H2AX foci. Cytogenetic analysis showed mainly numerical rather than structural chromosomal aberrations, indicating that ceramic particles were only weakly genotoxic to human cells in vitro compared to CoCr particles. Carrying these invstigations into the patient realm, biopsies from a pseudotumor following metal-on-metal hip resurfacing were analyzed using light and transmission electron microscopy (TEM), backscatter scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry. 42 Heavy macrophage infiltration was noted in black-pigmented specimens. Metal nanoparticles were observed exclusively within phagosomes of living macrophages and in fragments of dead macrophages. Although dead fibroblasts were seen juxtaposed with dead and disintegrated macrophages, the nanoparticles were not seen within either live or dead fibroblasts. Cr, but not Co, was the predominant component of the remaining wear nanoparticles in tissue. These results suggest that corrosion of Co in phagosomes of macrophages and resultant Co ion release led to tissue necrosis and adverse soft tissue reactions (pseudotumors). In another fibroblast model, 43 a differential display analysis on mRNA expression in the BALB3T3 A31-1-1 cells exposed to Co micro- or nanoparticles or Co ions revealed ten differentially expressed sequences representing candidate biomarkers for specific cellular responses. Treatment with Co nanoparticles was found to activate cellular defense and repair mechanisms. Further studies in BALB/3T3 cells 44 compared the cytotoxicity, genotoxicity and morphological transforming activity of Co nanoparticles and Co ions. The results revealed dose-dependent cytotoxicity for both compounds, as assessed by a colony forming unit (CFU) assay, that was initially higher for the Co nanoparticles than for Co ions, but which overlapped by day 3. The nanoparticles were internalized more efficiently than the Co ions. Co nanoparticles were positive in the micronucleus test and the comet assay, whereas Co ions were positive only with the latter. A significant increase in the formation of type III foci (morphologically transformed colonies) was observed only with the Co nanoparticles. The toxicological effects of Co nanoparticle aggregates were compared with those of Co ions using six different cell lines representing lung, liver, kidney, intestine, and the immune system. 45 Data analysis and predictive data modeling were conducted by employing a decision tree model. Concentration was determined to be the highest rank parameter, and the second-rank parameter was either the compound type (Co nanoparticles vs. ions) or the cell model, depending on the

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

concentration range; the lowest rank in the model was exposure duration. These Investigators believed that the toxic effects of aggregated Co nanoparticles were primarily due to Co ion dissolution from the particles. An earlier study evaluated the effects of Co nano- vs. microparticles on the production of several cytokines by peripheral blood mononuclear cells. 46 Co microparticles showed an inhibitory effect at all concentrations and towards all cytokines, whereas Co chloride solutions selectively inhibited Interleukin (IL)-2, IL-10 and tumor necrosis factor (TNF)-α at maximal concentration. Co nanoparticles induced an increase of TNF-α and interferon (IFN)-γ release and an inhibition of IL-10 and IL2: a cytokine pattern similar to that detected in the experimental and clinical autoimmunity. These Investigators concluded that immune endpoints should also be determined in subjects exposed to Co nanoparticles. The human monocytic cell line, U937, was employed to compare the ability of nanoscale Co vs. nanoscale TiO2 to cause alteration of transcription and activity of MMPs. 47 Exposure of U937 cells to Co nanoparticles, but not to TiO2, at non-cytotoxic doses, resulted in ROS generation and upregulation of MMP-2 and MMP-9 mRNA expression; this was mirrored by dose- and time-dependent increases in pro-MMP-2 and pro-MMP-9 gelatinolytic activities in supernatants. ProMMP activity increases were inhibited by pre-treatment with ROS scavengers or inhibitors. The same pattern of induction was observed with TIMP-2 but not TIMP-1 in U937 cells. Pretreatment with AP-1 inhibitor, curcumin, or the protein tyrosine kinase (PTK) specific inhibitor, herbimycin A or genistein, prior to exposure to Co nanoparticles significantly abolished induction of pro-MMP-2 and-9 activity. Thus, it was concluded that Co nanoparticles caused an imbalance between the expression and activity of MMPs and their inhibitors, due to oxidative stress. Using human lung epithelial cells, the intracellular oxidative effects following Co-containing silica nanoparticle exposure and the corresponding Co oxide were investigated. 48 The particles entered the cells efficiently and induced up to eightfold higher oxidative stress compared to cultures exposed to aqueous solutions of the metal. A similar “piggy-back” effect was proposed as one explanation of the toxicity of residual Co ions as components of selected nanotube preparations. 49 Cytotoxicity and oxidative stress responses of carbon and TiO2 nanomaterials in rainbow trout primary hepatocytes were determined, including C60 fullerenes, multiwall nanotubes (MWNT), single-wall nanotubes, and TiO2 of 5 and 200 nm in size. The nanoparticle agglomerates were cytotoxic at nominal concentrations, and certain nanotube preparations produced significant intracellular ROS production as well as cytotoxicity. Analyses of the reactive MWNT responsible for ROS production and cytotoxicity demonstrated the presence of residual Co, which was not present in the nonreactive/nonbioactive MWNT. Co alone was not able to induce the observed effects, whereas coexposure with MWNT resulted in increased cytotoxicity, possibly due to facilitated uptake. An important study assessed whether Co nanoparticles were more genotoxic than Co ions 50 in human peripheral blood leukocytes (PBLs). Uptake of Co ions was minimal compared to baseline levels, whereas Co nanoparticles were efficiently internalized. The genotoxicity end points were the frequency

S41

of binucleated micronucleated (BNMN) cells and the percentage of tail DNA (% Tail DNA) fragmentation. Co ions induced increases in BNMN frequency, whereas the nanoparticles showed only minor changes. Conversely, the comet assay showed a statistically significant dose-related increase in % Tail DNA for Co nanoparticles, whereas Co ions did not induce significant effects. Another study also used fresh human specimens in addition to cell lines to evaluate toxicity, and revealed that the former may be more instructive. 51 The toxicity of seven nanoparticle formulations, including those composed of Co, toward primary cultures of hematopoietic progenitor bone marrow cells from healthy donors was determined by CFU assays. It was revealed that antimony oxide and Co nanoparticles were toxic. The embryotoxicity of Co ferrite and several types of Au nanoparticles were compared using the Embryonic Stem Cell Test, an in vitro assay that allows classification of substances as strongly, weakly or non-embryotoxic. 52 These Investigators also proposed a method to discriminate the embryotoxicity of agents within the weakly embryotoxic range. The ranking of ID50 values classified Co ferrite nanoparticles coated with Au and silanes as non-embryotoxic; the remaining nanoparticles were weakly embryotoxic in this rank order: Au salt (HAuCl4·3H2O) > Co ferrite salt (CoFe2O4) > Co ferrite coated with silanes (Si-CoFe) > gold nanoparticles coated with hyaluronic acid (HA-Au). The toxicity of tungsten carbide (WC) and Co-doped WC nanoparticles toward different human cell lines as well as rat neuronal and glial cells was investigated. 53 Electron microscopy demonstrated the presence of WC nanoparticles within the cells. The nanoparticles (145 nm diameter) were not acutely toxic, but cytotoxicity became evident with Co doping, the most sensitive targets being astrocytes and colon epithelial cells. Studies of WC particles were extended to an aquatic environmental model, the rainbow trout gill cell line, RTgill-W1. 54 Scanning electron microscopy and energy dispersive X-ray elemental analyses indicated uptake of both WC and WC–Co nanoparticles into RTgill-W1 cells was primarily restricted to the cytoplasm. Shortterm exposure led to significant cytotoxicity at the highest particle concentrations, irrespective of the particle type or exposure medium. Long-term exposures led to enhanced toxicity by the Co-doped nanoparticles. The composition of the exposure media influenced the toxicity of Co ions, which may dissolve from the WC–Co nanoparticles; however, the toxicity observed by ionic Co alone did not explain the toxicity of the WC–Co nanoparticles, suggesting that the combination of metallic Co and WC was the cause of the increased particle toxicity. Busch et al 55 investigated the impact of these particles, compared to Co ions, on global gene expression level in human keratinocytes (HaCaT) in vitro. WC nanoparticles exerted minimal effects at the transcriptomic level after either 3 h or 3 days of exposure. In contrast, Co-doped WC nanoparticles caused significant transcriptional changes that were similar to those provoked by CoCl2. However, the latter exerted even more pronounced changes in the transcription patterns. Gene set enrichment analyses revealed that the differentially expressed genes were related to hypoxia response (e.g., HIF1α), carbohydrate metabolism, endocrine pathways, and targets

S42

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

of several transcription factors. To elucidate the mechanisms of health hazards of WC nanoparticles doped with Co, effects of WC–Co particles on nuclear factor erythroid 2-related factor 2 (Nrf2) signaling were investigated in the JB6 cell line. 56 After a 5 h treatment, Nrf2 was released from cytoplasmic Keap1 and translocated into the nucleus. Nrf2 target gene activities, including glutathione S-transferase (GST) and NAD(P)H: quinone oxidoreductase 1 (NQO1), increased by 24–48 h after treatment. ROS were produced in these nanoparticletreated cells. Pretreatment of the cells with catalase, but not sodium formate, resulted in a significant inhibition of Nrf2 target gene activation. Mariani et al 57 evaluated real-time changes in cell metabolisms linked to nanoparticle exposure. Fibroblast cultures were exposed to Co ferrite nanoparticles and the results were compared to conventional in vitro assays. Microphysiometer measurements showed a Co ferrite cytotoxic effect, confirmed by a colony forming assay. The genotoxicity in mice to orally administered CdSe 5 nm quantum dots and CdSe doped with 1% Co ions of similar size were assessed. 58 Bone marrow and liver samples were collected after two and seven days of treatment. The results indicated that after two days of treatment, doped quantum dots were able to induce DNA damage, formation of micronuclei, and generation of the DNA adduct, 8-hydroxy-2-deoxyguanosine. Increasing DNA damage, frequency of micronuclei formation and generation of DNA adducts were all observed with both the undoped and doped dots at higher doses and after seven days of treatment. These Investigators attributed to Co the potential to cause indirect in vivo genetic damage, via free radical-induced oxidative stress.

Iron-based nanoparticles By far the most activity has focused on Fe-based nanoparticles of the three elements ferromagnetic under physiological conditions, likely due to its superior magnetic susceptibility (218 and 90 emu/g for elemental Fe and Fe3O4, respectively), as well as the large natural reservoir of iron in the body, suggesting the comparative absence of toxicity of this element. Given the explosion of interest over the last few years in fabrication of new Fe-based nanoparticles, this review will confine itself to recent developments that have matured at least to in vivo studies of localization and also to anti-tumor activity. Localization The lymphatic biodistribution of superparamagnetic nanoparticle ferumoxtran, AMI 227/Sinerem™/Combidex™ in rats was characterized to elucidate the mechanism of uptake. 59 After catheterization of the thoracic lymph duct and i.v. injection of ferumoxtran, high concentrations of nanoparticles were found by MR relaxometry and atomic absorption spectroscopy (AAS) in the thoracic lymph within 90 min. No particles were found in the lymph cells, indicating that ferumoxtran was extracellular. The maximum concentration was reached later (12 h) in all node groups and then plateaued. The transcapillary pathway and

subsequent lymph drainage of the particles appeared to be major pathways for the delivery to the lymph nodes. The biodistribution of DOX conjugates of magnetite nanoparticles was assessed in mice following i.v. injection. 60 Electron spin resonance (ESR) analysis demonstrated that the DOX-nanoparticles substantially decreased DOX bioavailability in the heart and kidney compared to free DOX. A magnetic field of 210 mT and gradient of 200 mT/cm was effective in increasing the DOX-nanoparticle bioavailability at a target site, and to reduce hepatic clearance, resulting in the increased plasma bioavailability. The response was attributed to in vivo inhibition of phagocytic cells by the magnetic fields. The biodistribution of magnetite MNPs in ICR mice tissues was established using AAS. 61 They were found widely distributed in tissues including the heart, liver, spleen, lungs, kidneys, brain, stomach, small intestine, and bone marrow, with the majority in the liver and spleen. Fe3O4 MNP levels in brain tissue were higher in the MNP-treated group than in the controls, indicating penetration of the blood-brain barrier (BBB). Our groups are developing a magnetic vectoring system with more complex external magnetic fields to target a tumor and enhance tumor extravasation of systemically administered magnetite-based, silica-coated MNP (SiMNP) prodrug constructs. 62 Such localization has already been achieved as validated by MRI and SEM of tumor tissues in human ovarian and breast carcinoma xenograft models. 63,64 Superparamagnetic Fe3O4 poly e-caprolactone (PCL) nanoparticles (∼165 nm) were prepared with magnetizations of ∼10.2 emu/g. 65 Improved pharmacokinetic behavior and the potential for enhanced anti-tumor effects were examined in nude mice with s.c. xenografts of human HPAC pancreatic adenocarcinoma cells. These particles were loaded with gemcitabine and magnetic targeting manifested 15-fold higher intra-tumoral drug levels compared to administration of free gemcitabine. Block copolymer-coated magnetite nanoparticles were prepared for pancreatic cancer imaging by means of a chelation between the carboxylic acid groups in poly(ethylene glycol)-poly(aspartic acid) block copolymer and Fe on the surface of the iron oxide nanoparticles. 66 These nanoparticles were potent contrast agents for enhanced MR imaging of pancreatic cancer xenografts of the human BxPC3 cell line in nude mice. Iron staining of tumor tissue confirmed the nanoparticle accumulation. Silica-coated iron–carbon composite nanoparticles (200– 300 nm) were prepared to exploit the chemadsorptive properties of activated carbon for drug loading and the magnetic Fe for magnetic targeting. 67 99Tc-adsorbed composite particles showed marked biodistribution in the left hepatic lobe of pigs under external magnetic field influence. With DOX-loaded particles, the DOX content of hepatic tissue was ∼24-fold higher in the magnetically targeted left lobe than that in the control, nontargeted right lobe following intra-arterial infusion. These Investigators suggested that the nanoparticles penetrated through the capillary wall around the tissue interstitium and hepatic cells under the external magnetic force. MgFe2O4 magnetic nanoparticle composed of As2O3 (As2O3-MNPs) were prepared and their magnetic targeting in vivo was evaluated. 68 The 110 nm particles had a specific saturation magnetism of 8.65 emu/g. In vivo, the concentration of As2O3–MNPs in the liver was significantly

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

higher than that in the non-magnetically vectored group, whereas the concentration in the kidney was lower than that in the nonmagnetic group. The concentration in liver tissue in the magnetic group was 30.65 μg/g, more than four-fold the concentration in the same group in kidney tissue and ∼3-fold the concentration of drug in the liver tissue of the non-magnetic group. The utility of 100 nm iron oxide nanoparticles (saturation magnetization of 94 emu/g Fe) as a drug delivery platform for MRI-monitored magnetic targeting of brain tumors was evaluated. 69 Rats bearing orthotopic 9 L-gliosarcomas were injected i.v. with nanoparticles under a magnetic field density of 0.4 T that was applied for 30 min. MR images were acquired prior to administration of nanoparticles and immediately after magnetic targeting at 1 h intervals. MR image analysis revealed a five-fold increase in the total glioma uptake of nanoparticles compared to non-targeted tumors, which constituted a 3.6-fold enhancement in the target selectivity of nanoparticle accumulation in glioma over normal brain tissue. Later, this group evaluated polyethyleneimine (PEI)-modified magnetic nanoparticles, also with high saturation magnetization (93 emu/g Fe) for magnetic targeting to brain tumors. 70 Intra-carotid administration in conjunction with magnetic targeting resulted in a 30-fold increase in tumor accumulation compared to that observed with i.v. administration. Further, magnetic accumulation of cationic nanoparticles in tumors was ∼5-fold higher than that achieved with slightly anionic nanoparticles following intra-carotid administration, while there was no significant difference between the two in the contra-lateral brain. Gum Arabic-coated magnetic iron oxide nanoparticles (GA-MNP; 100 nm) were prepared with a saturation magnetization of 93.1 emu/g Fe and displayed superparamagnetic behavior. 71 GA coating enhanced colloidal stability and provided sites for functionalization. Based on in vitro characteristics, it was proposed that GA-MNP might be utilized as MRI-visible drug carriers for the purpose of magnetic tumor targeting. MR images post-vectoring confirmed tumor accumulation of GA-MNP following i.v. administration to rats with 9 L glioma xenografts and the application of an external magnetic field. ESR spectroscopy revealed a 12-fold increase in GA-MNP tumor accumulation compared to contralateral normal brain. The biodistribution patterns of polyethyleneglycol (PEG)MNPs in clearance organs and evaluation of proof-of-concept for their magnetic brain tumor targeting were reported. 72 Reductions in liver and spleen localization compared to parent starch-coated MNPs were observed; however, spleen accumulation increased at later time points. Enhanced magnetic brain tumor targeting of PEG-MNPs was confirmed in 9 L-glioma tumors, with up to 1.0% injected dose/g tissue nanoparticle delivery achieved. MRI and histological analyses both confirmed enhanced targeting. Fe2O3 magnetic nanoparticles (MNP) of 43 nm diameter were coated with the thermoresponsive polymer poly-n-isopropylacrylamide and then loaded with DOX. 73 In vivo magnetic targeting of DOX-loaded MNP to Buffalo rat hepatocellular carcinoma was validated by MRI and histology, in conjunction with in vitro evidence for hyperthermia-induced DOX release, supporting possible future applications in multi-modal treatment of cancer. Mesocrystalline CaCO3 particles encapsulating DOX, AuDNA, and magnetite nanoparticles were synthesized as stage 1

S43

microparticles (S1MPs), intended to marginate to vascular walls, after which the Au-DNA nanoparticles (stage 2 nanoparticles; S2NPs) and DOX would be released from S1MPs. 74 In a mouse tumor model, the targeted delivery of S2NPs into tumors was 10fold more efficient than that of the nanoparticles themselves, with DOX being widely distributed in tumor slices. Paclitaxel-loaded, superparamagnetic magnetite- and quantum dot-embedded polystyrene nanoparticles were conjugated to anti-prostate specific membrane antigen (anti-PSMA) antibodies. 75 Following i.v. injection into tumor-bearing nude mice, significant differences in fluorescent signals were observed between tumor regions treated with the PSMA-targeted nanocarrier system and the non-targeted nanocarrier system. DOX and iron oxide nanoparticles were loaded into human serum albumin (HSA) matrices. 76 The 50 nm nanoparticles caused the translocation of DOX across cell membranes (similar to the mechanism proposed for Abraxane™), followed by nuclear accumulation. In vivo, they retained tumor targeting capability as determined by both MRI and immunostaining. In the 4T1 breast cancer model, the DOX-loaded nanoparticles showed marked tumor suppression, comparable to that of Doxil and superior to free DOX. Anti-tumor activity The earliest and most compelling successful pre-clinical application of Fe-based nanoparticle delivery almost certainly should be credited to Alexiou et al, who established initial parameters for targeted magnetic drug delivery using intratumor arterial (i.a.) administration. 77-86 They used external magnetic fields to cause i.a.-administered ferrofluids, containing mitoxantrone (MTX)-loaded 100 nm nanoparticles, to accumulate at rabbit hind limb VX2 tumors fed by this artery. The colloidal ferrofluids were formed from iron oxides and hydroxides to produce aggregates of nanoparticles encapsulated within a starch polymer matrix, providing biological stabilization and sites for chemoabsorptive/electrostatic binding to MTX. Darkened tumor blood vessels were histologically evident immediately after treatment, with brown–black particles being distributed throughout the entire tumor. After the threemonth observation period, no viable tumor tissue was histologically evident in rabbits treated with the combination of i.a. administration and magnetic vectoring. In contrast, systemic (i.v.) administration failed, possibly due either to extensive nanoparticle clearance prior to reaching the tumor, or to premature MTX release prior to carrier extravasation. Translation of this ground-breaking approach to the clinic for treatment of head and neck cancer patients is underway. 87 The anionic glycosaminoglycan, dermatan sulfate (DS), was used to prepare an MR imaging agent composed of selfassembling, 5 nm nanoparticles of Fe 3+:deferoxamine (Fe:Df). 88 After i.v. injection, Fe:Df–DS rapidly targeted the neovascular endothelium of Dunning prostate R3327 AT1 rat tumors. Following endothelial transcytosis, it released from the abluminal surface to penetrate the interstitium from its initial site of high uptake in the well-perfused outer rim into the poorly perfused central region of the tumor. DS-DOX was prepared as 11 nm nanoparticles of DOX cores coated with DS. Analysis of

S44

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

tumor sections after i.v. injection showed much higher overall tumor fluorescence and deeper matrix penetration for DS-DOX than free DOX, and also resulted in enhanced tumor–cell internalization and nuclear localization of the drug. Using MX-1 human breast tumor xenografts and treatment at MTDs, observed times for tumor growth to seven-fold the initial volume were ∼8 daysfor control, ∼26 days for free DOX, and ∼43 days for DSDOX; at 90-day, total tumor regressions were observed in 0/10, 0/10, and 4/10 for control, free DOX and DS-DOX groups, respectively. These results were achieved even without exploiting magnetic vectoring: simply passive nanoparticle transport. Magnetite nanoparticle loaded with daunorubicin (DNR) were shown to overcome multidrug-resistance (MDR) of K562n/VCR human erythroid leukemia cells in vitro. 89 Mice with parental K562-n and K562-n/VCR s.c. xenografts were divided into groups including control, free DNR, unloaded nanoparticles, and DNR-loaded nanoparticles with or without a magnetic field juxtaposed on the xenograft surface. For the K562-n/VCR model, the tumor volume was markedly lower in the latter two groups than any of the others. This was associated with significant fracture, cell necrosis and later fibrosis, and reduced transcription of mdr-1 gene in the two groups, although there was no difference in the expression of P-glycoprotein (P-gp) between these groups. Although the DNR-loaded nanoparticles suppressed the growth of the MDR K562-n/VCR tumor, they did not further enhance DNR efficacy on the parental K562-n tumor. Of note, as in the Ranney et al study, 88 they did not observe further improvement of the anti-tumor effects from the influence of the external magnet. Superparamagnetic iron oxide (SPIO) nanoparticles were modified with o-carboxymethyl chitosan (OCMCS) and folic acid (FA) to attempt to improve their biocompatibility and ability to target specific tumor cells, simultaneously evading the reticuloendothelial system (RES; 90 ). Covalent surface-modification of SPIOs with OCMCS significantly reduced their capture by macrophages in vitro. Conversely, FA-modification enhanced their uptake by FR-positive tumor cell lines, but not by other cells without the folate receptor (FR). MR imaging and tumor histological analysis demonstrated the FA–OCMCS–SPIO could target FR-positive tumor cells in vivo. Wei et al 91,92 used BCNU covalently linked to carboxyl side chains of hydrophilic polymer-coated Fe3O4-based MNPs. These ∼10–20 nm superparamagnetic BCNU prodrugs were used for therapy in a rat C6 glioma model. The BBB was permeabilized by focused ultrasound (US); after US treatment, a magnetic field was juxtaposed to the rat cranium for up to 24 h. Rats received either (1) no treatment, (2) BCNU–MNPs administered via the external jugular vein, (3) US treatment before MNP administration, or (4) the same treatments in the reverse order, or (5) a combined treatment of US before, and magnetic targeting after, particle injection. The combination of US followed by magnetic vectoring enhanced the delivery of BCNU-loaded MNP to the gliomas as established by plasma optical emission spectroscopy; in the efficacy arms, dramatic tumor shrinkage was verified by serial MRI and terminal histological analyses, and prolonged survival. This has been extended to ∼90 nm MNP and tumor responses were observed even without the US. The increased efficiency was proposed to result from increased stability of

MNP-BCNU, or the external magnetic field causing enhanced local BCNU concentrations in the gliomas; the mechanisms underlying the efficacy of MNP-BCNU may be receptormediated endocytosis, phagocytosis and/or passive leakage of across defects in the BBB, enhanced by magnetic vectoring. Toxicology-in vitro studies Because endothelial cells (EC) are directly exposed to i.v.injected substances, characterization of effects on their physiology is fundamental to understanding possible host toxicity from nanoparticle administration. Human aortic endothelial cells (HAECs) were incubated in vitro with nanoparticles composed of Fe2O3, Y2O3, or ZnO; subsequently, mRNA and protein levels of Intercellular Adhesion Molecule-1 (ICAM-1), Interleukin-8 (IL-8), and Monocyte Chemoattractant Protein-1 (MCP-1) were measured, and nanoparticle interactions with HAECs were assessed by inductively coupled plasma mass spectrometry and TEM. 93 All three types of particles were internalized by HAECs and localized to intracellular vesicles; however, Fe2O3 nanoparticles did not induce an inflammatory response, whereas Y2O3 and ZnO nanoparticles did so. The toxicity and associated mechanisms of Fe2O3 and Fe3O4 nanoparticle exposure towards human ECV304 umbilical EC were investigated. 94 Both types of nanoparticles generated oxidative stress as well as increased nitric oxide (NO) production in ECV304 cells. Loss of mitochondrial membrane potential and nuclear chromatin condensation were early signs of apoptosis. Porcine aortic EC were exposed to Fe2O3 nanoparticles (20–40 nm) and alterations in ROS formation, morphology and cytoskeletal organization, death, and elastic modulus were assessed. 95 Intracellular ROS increased more than eight-fold after brief nanoparticle exposure, and EC doubled their initial length shortly thereafter, along with formation of actin stress fibers. When ROS formation was blocked pharmacologically, the cell morphology and actin cytoskeleton were unperturbed, and cell viability was protected. These Investigators suggest that if ROS formation is decreased using ROS inhibitors in vivo, higher nanoparticle concentrations might be used with greater efficacy and diminished side effects. Fe2O3 and Fe3O4 MNPs were assessed for their effects on HAECs and human U937 monocytic cells. 96 HAECs and U937 cells were exposed to a concentration range of 22 nm Fe 3 O 4 to 43 nm Fe 2 O 3 nanoparticles. Cytoplasmic vacuolation, mitochondrial swelling and cell death were induced in HAEC, along with an increase in NO production and NO synthesis (NOS) activity. Adhesion of monocytes to the HAECs was significantly enhanced as a consequence of the up-regulation of ICAM-1 and IL-8 expression. Phagocytosis and dissolution of nanoparticles by U937 cells were found to simultaneously provoke oxidative stress and mediate severe EC toxicity. Thus, i.v. administered iron oxide nanoparticles may induce endothelial inflammation and dysfunction via nanoparticle escape from phagocytosis and direct interaction with the endothelial layer, nanoparticle phagocytosis by monocytes followed by their dissolution, with subsequent effects of free iron ions, or nanoparticle phagocytosis to induce oxidative stress responses. Polyol-produced maghemite γ-

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

Fe2O3 nanoparticles were efficiently internalized by human EC in vitro, and caused cell death within 24 h of exposure, most likely through oxidative stress. 97 Although these nanoparticles were rapidly cleared through the urine, they led to toxicity in the liver, kidneys and lungs, while the brain and heart remain unaffected. These Investigators suggested that surface coating, cellular targeting, and local exposure should be considered before developing clinical applications. Dimercaptosuccinic acid (DMSA)-coated Fe nanoparticles (∼60 nm) were investigated for their uptake by cultured brain astrocytes. 98 Time- and concentration-dependent accumulation of cellular Fe was observed by EM, but without any cell toxicity. After 4 h of incubation with 100–4000 μM Fe equivalent nanoparticles, the cellular iron content reached levels between 200 and 2000 nmol/mg. Other studies have reported toxicity following intracellular delivery of Fe2O3 nanoparticles that adversely affected cell function. For example, such MNPs diminished the viability and capacity of PC12 cells to extend neurites in response to nerve growth factor (NGF 99). Ferritin contains 7 nm iron (ferrous) particles and a protein shell. Rat brain synaptosomes were used to investigate ferritin effects on uptake and release of glutamate, as well as free radical formation. 100 High concentrations of ferritin failed to induce spontaneous glutamate release, but did inhibit uptake. Ferritin also induced intrasynaptosomal ROS formation that was insensitive to the inhibitor of NADPH oxidase, DPI, and to CCCP, a mitochondrial uncoupler. These effects were proposed as potentially leading to neurodegeneration. Concerns over SPIOs from their long-term toxicity related to the production of toxic free iron during their biodegradation prompted an EMbased study. 101 SPIOs were found to be degraded after internalization by macrophages, leading to the core iron being incorporated into the non-toxic iron-storing protein, ferritin. The possible toxicities of silica-coated iron oxide nanoparticles were compared to commercially available dextran-coated iron oxide nanoparticles. 102 Silica-coated particles were nontoxic to primary human monocyte-derived macrophages; however, toxicity of the smaller silica-coated nanoparticles (30 and 50 nm) was observed for primary monocyte-derived dendritic cells, but not for the similar size dextran-coated iron oxide nanoparticles. Secretion of pro-inflammatory cytokines was not observed following exposure. The silica-coated nanoparticles were internalized more effectively than the dextran-coated nanoparticles, through an active, actin cytoskeleton-dependent process. The toxicity of mixed engineered carbon black (ECB) and maghemite iron oxide (Fe2O3) nanoparticles in human A549 lung epithelial cells was investigated using mixed Fe2O3 and ECB, mixed Fe2O3 and ECB nanoparticles plus ascorbic acid, and mixed Fe2 O 3 and surface-oxidized ECB (ox-ECB) nanoparticles. 103 Fe2O3 and ECB (or ox-ECB) were cointernalized in intracellular vesicles, over time resulting in increases in the amount of acidified lysosomes. Uptake of Fe2O3 was unaffected by the presence of ECB. Significant oxidant production and loss of cell viability occurred in cells exposed to Fe2O3 plus ECB, but not in cells exposed to Fe2O3 plus ox-ECB or in cells exposed to Fe2O3 and ECB with the addition of ascorbic acid. Thus, exposure to mixed Fe2O3 and ECB

S45

nanoparticles produces oxidants via the surface reductive capability of ECB when colocalized in acidic cellular compartments; this oxidative stress may be mitigated by an antioxidant (ascorbic acid) or by treatment of the ECB to ox-ECB, decreasing its surface reductive capacity. The surface characteristics of γ-Fe2O3 nanoparticles responsible for production of highly reactive hydroxyl radicals via the superoxide-driven Fenton reaction were investigated. 104 Spin-trapping electron paramagnetic resonance (EPR) experiments attributed free radical production primarily to catalysis at the 20–40 nm diameter particle surface, as opposed to dissolved metal ions released by the particles. Strikingly, catalytic centers on the nanoparticle surface were at least 50-fold more effective than the dissolved Fe 3+ ions in terms of free radical production. Of note, passivating the surfaces with oleate or bovine serum albumin was ineffective in suppressing this production. SPIOs (30 nm) coated with Tween 80 were used to characterize oxidative stress mechanisms after exposing the murine macrophage J774 cell line to them. 105 Time- and concentration-dependent effects were observed, with 6 h of exposure to 300–500 μg/mL reducing cell viability to ∼60%, largely via apoptosis. Nanoparticle-induced cell death was associated with ROS formation, and damage paralleled lactate dehydrogenase release. Based on these results, the Investigators concluded that the lowest effective level of SPIOs should be used to avoid ROS-mediated cell injury and death. The potential toxicities of three forms of SPIO nanoparticles with different surface chemistries (COOH, unmodified, and NH2) were evaluated in vitro via comparisons of gene expression patterns in human heart (HCM), brain (BE-2-C), and kidney (293T) cell lines. 106 The gene alterations and hierarchical clustering revealed that SPIO–COOH particles altered genes associated with cell proliferation due to their ROS properties, although it was cell-type dependent. The SPIO–NH2–responsive genes were largely associated with cellular metabolism, whereas the gene expression effects of unmodified SPIO treatment were more global. Toxicology-in vivo studies The toxic effects of inhalation exposure to Fe2O3 and ZnO nanoparticles were assessed in Male Wistar rats 107; although this route is typically not of greatest relevance to contemporary drug delivery settings, future applications might use this route for certain lung cancers. In the Fe2O3-treated rats, Fe content in liver and lung was significantly increased when examined at 36 h. In the ZnO-treated group, Zn content in liver was significantly increased already by 12 h and further increased by 36 h. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), and lactate dehydrogenase (LDH) in both nanoparticle-exposed groups were significantly decreased compared to the untreated controls; in contrast, damage in liver and lung tissues was evident histopathologically. In a related study, SPIO-containing patches were implanted in rats that were studied histologically over six months. 108 Degradation assessment of the SPIO–albumin patch was performed in vivo using MRI for iron content

S46

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

Figure 2. Successful magnetic vectoring of therapeutics (requirements for proof of concept).

localization and biodistribution. No degradation or accumulation into the RES, outside the area of the patch, was detected by MRI, MRI relaxometry, or histology. TEM showed initial local uptake of SPIOs into macrophages and cells of the RES, but this was not associated with apoptosispositive staining. These Investigators concluded that implanted SPION patches were relatively inert with slow, progressive local degradation over the six-month period. High-resolution magic-angle-spinning proton NMR spectroscopy coupled with multivariate statistical analysis was used to establish metabolic consequences in tissues, including clearance organs, kidney, liver and spleen, following i.v. injection of SPIO. 109 Functional alterations in these clearance organs were observed in metabolic pathways, including energy, lipid, glucose, and amino acid metabolism. The metabolic fate and potential for toxicity of SPIO were somewhat linked to their surface chemistry and particle size. Fe3O4 particles of 10 nm, 50 nm, or 1 μm diameter were injected i.p. into rats at a dose of 500 mg/kg, three times a week for five weeks in order to assess the effects of particle size/surface area parameters on any resultant toxicity. 110 Histopathological examinations of spleen and liver tissues were conducted, blood iron was measured photometrically, and that of the liver and the spleen by AAS and EPR. Fe3O4 nanoparticles demonstrated higher systemic toxicity compared to the microparticles, but between the nanoscale formulations, there was no relationship between particle size and toxicity. I.v. iron product administration has been associated with risks of oxidative stress. The effects of ferumoxytol were compared with those of ferric carboxymaltose, low molecular weight iron dextran, and iron sucrose in the liver, kidneys and heart of normal rats. 111 Compared to iron sucrose and ferric carboxymaltose, low molecular weight iron dextran and ferumoxytol caused renal and hepatic damage, manifested as proteinuria and increased liver enzyme levels. Higher levels of oxidative stress were also indicated: higher levels of malondialdehyde, increased anti-oxidant enzyme activities, and reduction in the reduced:

oxidized glutathione ratio. Inflammatory markers were also higher with ferumoxytol and low molecular weight iron dextran rat groups than with iron sucrose and ferric carboxymaltose treatments. Ferumoxytol contains a component with a more positive reduction potential, possibly enhancing Fe-catalyzed formation of ROS underlying the observed effects. Only low molecular weight iron dextran induced oxidative stress and inflammation in the heart. The ocular toxicity of MNPs (50 nm) or 4 μm magnetic particles were assessed regarding effects on intraocular pressure (IOP), corneal endothelial cell count, retinal morphology, and photoreceptor function. 112 Sprague–Dawley rats were injected in one eye with either 50 nm or 4 μm magnetic particles, and an equal volume of PBS into the opposite eye. Electroretinograms (ERG) were used to determine if MNPs induced functional changes to the photoreceptor layers; enucleated eyes were sectioned for histology and immunofluorescence. Compared to controls, MNPs did not alter IOP measurements. ERG amplitudes for α-waves and β-waves were not significantly different between injected and non-injected eyes. Histological sectioning and immunofluorescence staining showed little difference in MNP-injected animals compared to controls. In contrast, corneal endothelial cell numbers were significantly lower in the 4 μm particle-injected eyes compared to either MNP- or PBS-injected eyes. Furthermore, iron deposition was detected after 4 μm particle injection, but not MNP injection. Overall, the data suggested that MNPs were safe for intraocular use. Mice given intranasal Fe2O3 nanoparticles showed pathological alterations in the olfactory bulb, hippocampus and striatum, and caused microglial proliferation, activation and recruitment to these structures. 113 BV2 microglial cells exposed to Fe2O3 nanoparticles responded with proliferation, phagocytosis and generation of ROS and NO; however, the nanoparticles did not induce inflammatory mediator (IL-1β, IL-6 and TNF-α) release. The iron redox state of USPION (ultrafine SPIO nanoparticles) was found to markedly affect their cellular uptake and

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50 114

subsequent induction of DNA damage. The generation of Fe ions, chromosomal damage due to DNA fragmentation, and accumulation of oxidized DNA bases lesions, supported a mechanism of oxidative DNA damage by γ-Fe2O3 dextrancoated USPION. The oxidation state of iron can vary considerably across the solid solution range of Fe3O4 to γ-Fe2O3 during the synthesis of USPION and subsequent environmental oxidation/reduction reactions. The resulting non-stoichiometric ratios of Fe 2+ and Fe 3+ may contribute to the varying redox potential and surface coordination chemistries and could underly the observed differential uptake, oxidative stress and the corresponding genotoxicity of USPION formulations.

Conclusions Development of targeted delivery of chemotherapeutic agents is a multi-disciplinary endeavor that includes the concept of using magnetic fields to direct MNP to the interstitial tumor microenvironment. Although the origins of this approach can be traced back more than three decades (reviewed in Ref [115]), the last several years have witnessed an enormous proliferation of studies using nanoparticles based on the three ferromagnetic elements: Co, Ni and Fe. Such vectors can either be used to mediate a hyperthermic effect, predominantly causing tumor necrosis, or to deliver drugs to achieve intra-tumoral levels greatly exceeding those observed with maximum tolerated doses of free drugs; the intent is to overcome pharmacologically-based therapeutic resistance, while reducing normal tissue toxicity. As these investigations pushed forward, at almost the same pace, toxicological evaluations of nanoparticles composed of these elements or their composite hybrid materials have emerged, reflecting the high level of scrutiny to which nanomaterials are subjected. In this review, we have attempted to define the current understanding of the effectiveness of ferromagnetic materials as magnetic delivery vehicles, across a matrix of performance parameters that forms the basis for a successful treatment modality. In Figure 2, we have captured an over-arching current snapshot of this matrix. Ni-based MNP platforms, being relatively low in magnetic susceptibility compared to Co and Fe and yet highly active in exerting toxicity effects, do not optimally meet the requirements for pursuing a clinically viable approach. Similarly, Co-based MNP systems, while demonstrating a greater magnetic susceptibility than that of Ni- and close to that of Fe-based MNP, also present significant toxic effects. In summary, Fe-based MNP, despite evidence that they are not as benign as the existence of an endogenous iron pool might initially suggest, appear to provide the best overall scope of performance properties. Further, the advanced stage of preclinical, human tumor xenograft studies with Fe-based MNP gives great confidence that the broader requirements for moving forward to clinical applicability will be met.

Contributors All authors contributed equally.

S47

Competing interests No competing interests.

Provenance and peer review Commissioned and externally peer reviewed.

References 1. Rodríguez-Llamazares S, Merchán J, Olmedo I, et al. Ni/Ni oxides nanoparticles with potential biomedical applications obtained by displacement of a nickel–organometallic complex. J Nanosci Nanotechnol 2008;8(8):3820-7. 2. Guo D, Wu C, Hu H, Wang X, Li X, Chen B. Study on the enhanced cellular uptake effect of daunorubicin on leukemia cells mediated via functionalized nickel nanoparticles. Biomed Mater 2009;4(2):025013. 3. Kale SN, Jadhav AD, Verma S, et al. Characterization of biocompatible NiCo2O4 nanoparticles for applications in hyperthermia and drug delivery. Nanomedicine 2012;8(4):452-9. 4. Gupta AK, Naregalkar RR, Vaidya VD, Gupta M. Recent advances on surface engineering of magnetic iron oxide nanoparticles and their biomedical applications. Nanomedicine (Lond) 2007;2(1):23-39 [Review]. 5. Rana S, Gallo A, Srivastava RS, Misra RD. On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: functionalization, conjugation and drug release kinetics. Acta Biomater 2007;3(2):233-42. 6. Ahamed M, Akhtar MJ, Siddiqui MA, et al. Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells. Toxicology 2011;283(2–3):101-8. 7. Lee SM, Lee OS, O’Halloran TV, Schatz GC, Nguyen ST. Triggered release of pharmacophores from Ni(HAsO3)-loaded polymer-caged nanobin enhances pro-apoptotic activity: a combined experimental and theoretical study. ACS Nano 2011;5(5):3961-9. 8. Chen H, Ahn R, Van den Bossche J, Thompson DH, O’Halloran TV. Folate-mediated intracellular drug delivery increases the anticancer efficacy of nanoparticulate formulation of arsenic trioxide. Mol Cancer Ther 2009;8(7):1955-63. 9. Feng B, Tomizawa K, Michiue H, et al. Delivery of sodium borocaptate to glioma cells using immunoliposome conjugated with anti-EGFR antibodies by ZZ-His. Biomaterials 2009;30(9):1746-55. 10. Benhabbour SR, Luft JC, Kim D, et al. In vitro and in vivo assessment of targeting lipid-based nanoparticles to the epidermal growth factor– receptor (EGFR) using a novel Heptameric Z(EGFR) domain. J Control Release 2012;158(1):63-71. 11. Morimoto Y, Hirohashi M, Ogami A, et al. Pulmonary toxicity following an intratracheal instillation of nickel oxide nanoparticle agglomerates. J Occup Health 2011;53(4):293-5. 12. Morimoto Y, Oyabu T, Ogami A, et al. Investigation of gene expression of MMP-2 and TIMP-2 mRNA in rat lung in inhaled nickel oxide and titanium dioxide nanoparticles. Ind Health 2011;49(3):344-52. 13. Kang GS, Gillespie PA, Gunnison A, Rengifo H, Koberstein J, Chen LC. Comparative pulmonary toxicity of inhaled nickel nanoparticles; role of deposited dose and solubility. Inhal Toxicol 2011;23(2):95-103. 14. Horie M, Fukui H, Nishio K, et al. Evaluation of acute oxidative stress induced by NiO nanoparticles in vivo and in vitro. J Occup Health 2011;53(2):64-74. 15. Cuevas AK, Liberda EN, Gillespie PA, Allina J, Chen LC. Inhaled nickel nanoparticles alter vascular reactivity in C57BL/6 mice. Inhal Toxicol 2010;22(Suppl 2):100-6. 16. Liberda EN, Cuevas AK, Gillespie PA, Grunig G, Qu Q, Chen LC. Exposure to inhaled nickel nanoparticles causes a reduction in number

S48

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32. 33.

34.

35.

36.

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50 and function of bone marrow endothelial progenitor cells. Inhal Toxicol 2010;22(Suppl 2):95-9. Kang GS, Gillespie PA, Gunnison A, Moreira AL, Tchou-Wong KM, Chen LC. Long-term inhalation exposure to nickel nanoparticles exacerbated atherosclerosis in a susceptible mouse model. Environ Health Perspect 2011;119(2):176-81. Parsons JG, Lopez ML, Gonzalez CM, Peralta-Videa JR, GardeaTorresdey JL. Toxicity and biotransformation of uncoated and coated nickel hydroxide nanoparticles on mesquite plants. Environ Toxicol Chem 2010;29(5):1146-54. Phillips JI, Green FY, Davies JC, Murray J. Pulmonary and systemic toxicity following exposure to nickel nanoparticles. Am J Ind Med 2010;53(8):763-7. Nishi K, Morimoto Y, Ogami A, et al. Expression of cytokine-induced neutrophilchemoattractantinratlungsbyintratrachealinstillationofnickel oxide nanoparticles. Inhal Toxicol 2009;21(12):1030-9. Ispas C, Andreescu D, Patel A, Goia DV, Andreescu S, Wallace KN. Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish. Environ Sci Technol 2009;43(16):6349-56. Ogami A, Morimoto Y, Myojo T, et al. Pathological features of different sizes of nickel oxide following intratracheal instillation in rats. Inhal Toxicol 2009;21(10):812-8. Griffitt RJ, Luo J, Gao J, Bonzongo JC, Barber DS. Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environ Toxicol Chem 2008;27(9):1972-8. Blaise C, Gagné F, Férard JF, Eullaffroy P. Ecotoxicity of selected nano-materials to aquatic organisms. Environ Toxicol 2008;23(5): 591-8. Pietruska JR, Liu X, Smith A, et al. Bioavailability, intracellular mobilization of nickel, and HIF-1α activation in human lung epithelial cells exposed to metallic nickel and nickel oxide nanoparticles. Toxicol Sci 2011;124(1):138-48. Morimoto Y, Ogami A, Todoroki M, et al. Expression of inflammationrelated cytokines following intratracheal instillation of nickel oxide nanoparticles. Nanotoxicology 2010;4(2):161-76. Cho WS, Duffin R, Poland CA, et al. Metal oxide nanoparticles induce unique inflammatory footprints in the lung: important implications for nanoparticle testing. Environ Health Perspect 2010;118(12):1699-706. Peters K, Unger RE, Gatti AM, Sabbioni E, Tsaryk R, Kirkpatrick CJ. Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro. Int J Immunopathol Pharmacol 2007;20(4):685-95. Monteiller C, Tran L, MacNee W, et al. The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area. Occup Environ Med 2007;64(9):609-15. Yoon TJ, Kim JS, Kim BG, Yu KN, Cho MH, Lee JK. Multifunctional nanoparticles possessing a magnetic motor effect for drug or gene delivery. Angew Chem Int Ed Engl 2005;44(7):1068-71. Baldi G, Bonacchi D, Franchini MC, et al. Synthesis and coating of cobalt ferrite nanoparticles: a first step toward the obtainment of new magnetic nanocarriers. Langmuir 2007;23(7):4026-8. Papis E, Rossi F, Raspanti M, et al. Engineered cobalt oxide nanoparticles readily enter cells. Toxicol Lett 2009;189(3):253-9. Withey AB, Chen G, Nguyen TL, Stenzel MH. Macromolecular cobalt carbonyl complexes encapsulated in a click-cross-linked micelle structure as a nanoparticle to deliver cobalt pharmaceuticals. Biomacromolecules 2009;10(12):3215-26. Sherlock SP, Tabakman SM, Xie L, Dai H. Photothermally enhanced drug delivery by ultrasmall multifunctional FeCo/graphitic shell nanocrystals. ACS Nano 2011;5(2):1505-12. Liu L, Zhang H, Meng X, Yin J, Li D, Liu C. Dinuclear metal(II) complexes of polybenzimidazole ligands as carriers for DNA delivery. Biomaterials 2010;31(6):1380-91. Wu H, Liu G, Wang X, et al. Solvothermal synthesis of cobalt ferrite nanoparticles loaded on multiwalled carbon nanotubes for magnetic

37.

38.

39.

40. 41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

resonance imaging and drug delivery. Acta Biomater 2011;7(9): 3496-504. Scarberry KE, Dickerson EB, McDonald JF, Zhang ZJ. Magnetic nanoparticle-peptide conjugates for in vitro and in vivo targeting and extraction of cancer cells. J Am Chem Soc 2008;130(31):10258-62. Pouponneau P, Leroux JC, Soulez G, Gaboury L, Martel S. Coencapsulation of magnetic nanoparticles and doxorubicin into biodegradable microcarriers for deep tissue targeting by vascular MRI navigation. Biomaterials 2011;32(13):3481-6. Papageorgiou I, Brown C, Schins R, et al. The effect of nano- and micron-sized particles of cobalt-chromium alloy on human fibroblasts in vitro. Biomaterials 2007;28(19):2946-58. Bhabra G, Sood A, Fisher B, et al. Nanoparticles can cause DNA damage across a cellular barrier. Nat Nanotechnol 2009;4(12):876-83. Tsaousi A, Jones E, Case CP. The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles. Mutat Res 2010; 697(1–2):1-9. Xia Z, Kwon YM, Mehmood S, Downing C, Jurkschat K, Murray DW. Characterization of metal-wear nanoparticles in pseudotumor following metal-on-metal hip resurfacing. Nanomedicine 2011. Papis E, Gornati R, Prati M, Ponti J, Sabbioni E, Bernardini G. Gene expression in nanotoxicology research: analysis by differential display in BALB3 T3 fibroblasts exposed to cobalt particles and ions. Toxicol Lett 2007;170(3):185-92. Ponti J, Sabbioni E, Munaro B, et al. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3 T3 mouse fibroblasts. Mutagenesis 2009;24(5):439-45. Horev-Azaria L, Kirkpatrick CJ, Korenstein R, et al. Predictive toxicology of cobalt nanoparticles and ions: comparative in vitro study of different cellular models using methods of knowledge discovery from data. Toxicol Sci 2011;122(2):489-501. Petrarca C, Perrone A, Verna N, et al. Cobalt nano-particles modulate cytokine in vitro release by human mononuclear cells mimicking autoimmune disease. Int J Immunopathol Pharmacol 2006;19(suppl 4):11-4. Wan R, Mo Y, Zhang X, Chien S, Tollerud DJ, Zhang Q. Matrix metalloproteinase-2 and -9 are induced differently by metal nanoparticles in human monocytes: the role of oxidative stress and protein tyrosine kinase activation. Toxicol Appl Pharmacol 2008;233(2):276-85. Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ Sci Technol 2007;41(11):4158-63. Thomas KV, Farkas J, Farmen E, et al. Effects of dispersed aggregates of carbon and titanium dioxide engineered nanoparticles on rainbow trout hepatocytes. J Toxicol Environ Health A 2011;74(7–9):466-77. Colognato R, Bonelli A, Ponti J, et al. Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro. Mutagenesis 2008;23(5):377-82. Bregoli L, Chiarini F, Gambarelli A, et al. Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines. Toxicology 2009;262(2):121-9. Di Guglielmo C, López DR, De Lapuente J, Mallafre JM, Suàrez MB. Embryotoxicity of cobalt ferrite and gold nanoparticles: a first in vitro approach. Reprod Toxicol 2010;30(2):271-6. Bastian S, Busch W, Kühnel D, et al. Toxicity of tungsten carbide and cobalt-doped tungsten carbide nanoparticles in mammalian cells in vitro. Environ Health Perspect 2009;117(4):530-6. Kühnel D, Busch W, Meissner T, et al. Agglomeration of tungsten carbide nanoparticles in exposure medium does not prevent uptake and toxicity toward a rainbow trout gill cell line. Aquat Toxicol 2009;93(2– 3):91-9. Busch W, Kühnel D, Schirmer K, Scholz S. Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes. BMC Genomics 2010(11):65.

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50 56. Zhang XD, Zhao J, Bowman L, Shi X, Castranova V, Ding M. Tungsten carbide–cobalt particles activate Nrf2 and its downstream target genes in JB6 cells possibly by ROS generation. J Environ Pathol Toxicol Oncol 2010;29(1):31-40. 57. Mariani V, Ponti J, Giudetti G, et al. Online monitoring of cell metabolism to assess the toxicity of nanoparticles: the case of cobalt ferrite. Nanotoxicology 2011. 58. Khalil WK, Girgis E, Emam AN, Mohamed MB, Rao KV. Genotoxicity evaluation of nanomaterials: DNA damage, micronuclei, and 8-hydroxy-2-deoxyguanosine induced by magnetic doped CdSe quantum dots in male mice. Chem Res Toxicol 2011;24(5): 640-50. 59. Réty F, Clément O, Siauve N, et al. MR lymphography using iron oxide nanoparticles in rats: pharmacokinetics in the lymphatic system after intravenous injection. J Magn Reson Imaging 2000;12(5):734-9. 60. Mykhaylyk OM, Dudchenko NO, Dudchenko AK. Pharmacokinetics of the doxorubicin magnetic nanoconjugate in mice Effects of the nonuniform stationary magnetic field. Ukr Biokhim Zh 2005; 77(5):80-92. 61. Wang J, Chen Y, Chen B, et al. nanoparticles in mice. Int J Nanomed 2010;5:861-6. 62. Lian G, Lewelling K, Johnson M, Dormer K, Gibson D, Seeney C. nanoparticles for biomedical applications. Microsc Microanal 2004;10(Suppl 2). 63. Klostergaard J, Bankson J, Auzenne E, Gibson D, Yuill W, Seeney C. Magnetic vectoring of magnetically responsive nanoparticles within the murine peritoneum. J Magn Magn Mater 2007;311(1): 330-5. 64. Klostergaard J, Bankson J, Woodward W, Gibson D, Seeney C. Magnetically responsive nanoparticles for vectored delivery of cancer therapeutics. AIP Conf Proc 2011;1311(1):382-7. 65. Gang J, Park SB, Hyung W, et al. and gemcitabine enhance anti-tumor effect in pancreatic cancer xenograft mouse model. J Drug Target 2007;15(6):445-53. 66. Kumagai M, Kano MR, Morishita Y, et al. by block copolymer-coated magnetite nanoparticles with TGF-beta inhibitor. J Control Release 2009;140(3):306-11. 67. Cao H, Gan J, Wang S, et al. Novel silica-coated iron–carbon composite particles and their targeting effect as a drug carrier. J Biomed Mater Res A 2008;86(3):671-7. 68. Yang GF, Li XH, Zhao Z, Wang WB. studies of arsenic trioxide Mg– Fe ferrite magnetic nanoparticles. Acta Pharmacol Sin 2009;30(12): 1688-93. 69. Chertok B, Moffat BA, David AE, et al. Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 2008;29(4):487-96. 70. Chertok B, David AE, Yang VC. Polyethyleneimine-modified iron oxide nanoparticles for brain tumor drug delivery using magnetic targeting and intra-carotid administration. Biomaterials 2010; 31(24):6317-24. 71. Zhang L, Yu F, Cole AJ, et al. Gum arabic-coated magnetic nanoparticles for potential application in simultaneous magnetic targeting and tumor imaging. AAPS J 2009;11(4):693-9. 72. Cole AJ, David AE, Wang J, Galbán CJ, Yang VC. Magnetic brain tumor targeting and biodistribution of long-circulating PEG-modified, cross-linked starch-coated iron oxide nanoparticles. Biomaterials 2011;32(26):6291-301. 73. Purushotham S, Chang PE, Rumpel H, et al. Thermoresponsive coreshell magnetic nanoparticles for combined modalities of cancer therapy. Nanotechnology 2009;20(30):305101. 74. Zhao Y, Lu Y, Hu Y, et al. mesocrystals for multistage delivery in cancer therapy. Small 2010;6(21):2436-42. 75. Cho HS, Dong Z, Pauletti GM, et al. Fluorescent, superparamagnetic nanospheres for drug storage, targeting, and imaging: a multifunctional nanocarrier system for cancer diagnosis and treatment. ACS Nano 2010;4(9):5398-404.

S49

76. Quan Q, Xie J, Gao H, et al. HSA coated iron oxide nanoparticles as drug delivery vehicles for cancer therapy. Mol Pharm 2011;8(5): 1669-76. 77. Alexiou C, Arnold W, Klein RJ, et al. Locoregional cancer treatment with magnetic drug targeting. Cancer Res 2000;60(23):6641-8. 78. Lübbe AS, Alexiou C, Bergemann C. Clinical applications of magnetic drug targeting. J Surg Res 2001;95(2):200-6 Review. 79. Alexiou C, Jurgons R, Schmid RJ, et al. Magnetic drug targeting – biodistribution of the magnetic carrier and the chemotherapeutic agent mitoxantrone after locoregional cancer treatment. J Drug Target 2003; 11(3):139-49. 80. Alexiou C, Schmid RJ, Jurgons R, et al. Targeting cancer cells: magnetic nanoparticles as drug carriers. Eur Biophys J 2006;35(5): 446-50. 81. Wiekhorst F, Seliger C, Jurgons R, et al. Quantification of magnetic nanoparticles by magnetorelaxometry and comparison to histology after magnetic drug targeting. J Nanosci Nanotechnol 2006;6(9–10): 3222-5. 82. Alexiou C, Jurgons R, Seliger C, Brunke O, Iro H, Odenbach S. Delivery of superparamagnetic nanoparticles for local chemotherapy after intraarterial infusion and magnetic drug targeting. Anticancer Res 2007;27(4A):2019-22. 83. Richter H, Wiekhorst F, Schwarz K, et al. Magnetorelaxometric quantification of magnetic nanoparticles in an artery model after ex vivo magnetic drug targeting. Phys Med Biol 2009;54(18):N417-24. 84. Tietze R, Schreiber E, Lyer S, Alexiou C. Mitoxantrone loaded superparamagnetic nanoparticles for drug targeting: a versatile and sensitive method for quantification of drug enrichment in rabbit tissues using HPLC–UV. J Biomed Biotechnol 2010;2010:597304. 85. Lyer S, Tietze R, Jurgons R, et al. Visualisation of tumour regression after local chemotherapy with magnetic nanoparticles – a pilot study. Anticancer Res 2010;30(5):1553-7. 86. Tietze R, Rahn H, Lyer S, et al. Visualization of superparamagnetic nanoparticles in vascular tissue using XμCT and histology. Histochem Cell Biol 2011;135(2):153-8. 87. El-Sayed IH. Nanotechnology in head and neck cancer: the race is on. Curr Oncol Rep 2010;12(2):121-8. 88. Ranney D, Antich P, Dadey E, et al. Dermatan carriers for neovascular transport targeting, deep tumor penetration and improved therapy. J Control Release 2005;109(December (1–3)):222-35. 89. Lai BB, Chen BA, Cheng J, et al. greatly enhance the responses of multidrug-resistant K562 leukemic cells in a nude mouse xenograft model to chemotherapy. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2009; 17(2):345-51. 90. Fan C, Gao W, Chen Z, et al. Tumor selectivity of stealth multifunctionalized superparamagnetic iron oxide nanoparticles. Int J Pharm 2011;404(1–2):180-90. 91. Chen PY, Liu HL, Hua MY, et al. Novel magnetic/ultrasound focusing system enhances nanoparticle drug delivery for glioma treatment. Neuro Oncol 2010;12(10):1050-60. 92. Hua MY, Liu HL, Yang HW, et al. The effectiveness of a magnetic nanoparticle-based delivery system for BCNU in the treatment of gliomas. Biomaterials 2011;32(2):516-27. 93. Gojova A, Guo B, Kota RS, Rutledge JC, Kennedy IM, Barakat AI. Induction of inflammation in vascular endothelial cells by metal oxide nanoparticles: effect of particle composition. Environ Health Perspect 2007;115(3):403-9. 94. Zhu MT, Wang Y, Feng WY, et al. Oxidative stress and apoptosis induced by iron oxide nanoparticles in cultured human umbilical endothelial cells. J Nanosci Nanotechnol 2010;10(12):8584-90. 95. Buyukhatipoglu K, Clyne AM. Superparamagnetic iron oxide nanoparticles change endothelial cell morphology and mechanics via reactive oxygen species formation. J Biomed Mater Res A 2010. 96. Zhu MT, Wang B, Wang Y, et al. Endothelial dysfunction and inflammation induced by iron oxide nanoparticle exposure: risk factors for early atherosclerosis. Toxicol Lett 2011;203(2):162-71.

S50

J. Klostergaard, C.E. Seeney / Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) S37–S50

97. Hanini A, Schmitt A, Kacem K, Chau F, Ammar S, Gavard J. Evaluation of iron oxide nanoparticle biocompatibility. Int J Nanomed 2011;6:787-94. 98. Geppert M, Hohnholt MC, Thiel K, et al. Uptake of dimercaptosuccinate-coated magnetic iron oxide nanoparticles by cultured brain astrocytes. Nanotechnology 2011;22(14):145101. 99. Pisanic 2nd TR, Blackwell JD, Shubayev VI, Fiñones RR, Jin S. Nanotoxicity of iron oxide nanoparticle internalization in growing neurons. Biomaterials 2007;28(16):2572-81. 100. Alekseenko AV, Waseem TV, Fedorovich SV. Ferritin, a protein containing iron nanoparticles, induces reactive oxygen species formation and inhibits glutamate uptake in rat brain synaptosomes. Brain Res 2008;(1241):193-200. 101. López-Castro JD, Maraloiu AV, Delgado JJ, et al. From synthetic to natural nanoparticles: monitoring the biodegradation of SPIO (P904) into ferritin by electron microscopy. Nanoscale 2011;3(11):4597-9. 102. Kunzmann A, Andersson B, Vogt C, et al. Efficient internalization of silica-coated iron oxide nanoparticles of different sizes by primary human macrophages and dendritic cells. Toxicol Appl Pharmacol 2011;253(2):81-93. 103. Berg JM, Ho S, Hwang W, et al. Internalization of carbon black and maghemite iron oxide nanoparticle mixtures leads to oxidant production. Chem Res Toxicol 2010 November 10. 104. Voinov MA, Sosa Pagán JO, Morrison E, Smirnova TI, Smirnov AI. Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity. J Am Chem Soc 2011;133(1):35-41. 105. Naqvi S, Samim M, Abdin M, et al. Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress. Int J Nanomed 2010;5:983-9. 106. Mahmoudi M, Laurent S, Shokrgozar MA, Hosseinkhani M. Toxicity evaluations of superparamagnetic iron oxide nanoparticles: cell vision

107.

108.

109.

110.

111.

112.

113.

114.

115.

versus physicochemical properties of nanoparticles. ACS Nano 2011;5(9):7263-76. Wang L, Wang L, Ding W, Zhang F. Acute toxicity of ferric oxide and zinc oxide nanoparticles in rats. J Nanosci Nanotechnol 2010; 10(12):8617-24. Schlachter EK, Widmer HR, Bregy A, et al. Metabolic pathway and distribution of superparamagnetic iron oxide nanoparticles: in vivo study. Int J Nanomed 2011;6:1793-800. Feng J, Liu H, Bhakoo KK, Lu L, Chen Z. A metabonomic analysis of organ specific response to USPIO administration. Biomaterials 2011;32(27):6558-69. Katsnelson BA, Degtyareva TD, Minigalieva II, et al. Subchronic systemic toxicity and bioaccumulation of Fe3O4 nano- and microparticles following repeated intraperitoneal administration to rats. Int J Toxicol 2011;30(1):59-68. Toblli JE, Cao G, Oliveri L, Angerosa M. Assessment of the extent of oxidative stress induced by intravenous ferumoxytol, ferric carboxymaltose, iron sucrose and iron dextran in a nonclinical model. Arzneimittelforschung 2011;61(7):399-410. Raju HB, Hu Y, Vedula A, Dubovy SR, Goldberg JL. Evaluation of magnetic micro- and nanoparticle toxicity to ocular tissues. PLoS One 2011;6(5):e17452. Wang Y, Wang B, Zhu MT, et al. Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure. Toxicol Lett 2011;205(1):26-37. Singh N, Jenkins GJ, Nelson BC, et al. The role of iron redox state in the genotoxicity of ultrafine superparamagnetic iron oxide nanoparticles. Biomaterials 2012;33(1):163-70. Seeney C, Ojwang JO, Weiss RD, Klostergaard J. Magnetically vectored platforms for the targeted delivery of therapeutics to tumors: history and current status. Nanomedicine (Lond) 2012;7(2):289-99.