Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging��� Omid Veiseh, Jonathan W. Gunn, Miqin Zhang ��� Department of Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA a b s t r a c t a r t i c l e i n f o Article history: Received 31 December 2008 Accepted 17 October 2009 Available online 10 November 2009 Keywords: Magnetic nanoparticle Molecular targeting MRI Contrast agents Gene therapy Drug release Bioconjugation Biological barriers Blood Brain Barrier Surface modification Physicochemical properties Magnetic nanoparticles (MNPs) represent a class of non-invasive imaging agents that have been developed for magnetic resonance (MR) imaging. These MNPs have traditionally been used for disease imaging via passive targeting, but recent advances have opened the door to cellular-specific targeting, drug delivery, and multi-modal imaging by these nanoparticles. As more elaborate MNPs are envisioned, adherence to proper design criteria (e.g. size, coating, molecular functionalization) becomes even more essential. This review summarizes the design parameters that affect MNP performance in vivo, including the physicochemical properties and nanoparticle surface modifications, such as MNP coating and targeting ligand functionaliza- tions that can enhance MNP management of biological barriers. A careful review of the chemistries used to modify the surfaces of MNPs is also given, with attention paid to optimizing the activity of bound ligands while maintaining favorable physicochemical properties. �� 2009 Elsevier B.V. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2. Nanoparticle design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2.1. In vivo barriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 2.2. Physicochemical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.2.1. Hydrodynamic Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.2.2. Shape. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.2.3. Surface properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.3. Directing nanoparticles in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286 2.4. Drug loading and Release. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 2.5. Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 3. Fabrication of target-specific magnetic nanoparticles (MNPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 3.1. MNP core fabrication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 3.2. Coating of SPIONs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 3.3. Organic surface coatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 3.3.1. Poly(ethylene glycol) PEG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 3.3.2. Dextran. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 3.3.3. Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 3.3.4. Polyethyleneimine (PEI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 3.3.5. Liposomes and Micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292 3.3.6. Copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Advanced Drug Delivery Reviews 62 (2010) 284���304 ��� This review is part of the Advanced Drug Delivery Reviews theme issue on ���Targeted Delivery Using Inorganic Nanosystem���. ��� Correspondingauthor.DepartmentofMaterials Science&Engineering,302LRobertsHall,Universityof Washington,Seattle,WA98195-2120.Tel.:+12066169356 fax:+12065433100. E-mail address: mzhang@u.washington.edu (M. Zhang). 0169-409X/$ ��� see front matter �� 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.11.002 Contents lists available at ScienceDirect Advanced Drug Delivery Reviews journal homepage: www.elsevier.com/locate/addr

3.4. Surface modification chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 3.4.1. Covalent linkages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 3.4.2. Physical interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 3.5. SPION targeting strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295 4. MNP drug delivery vehicles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4.1. Chemotherapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4.2. Radiotherapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4.3. Biotherapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4.3.1. Therapeutic peptides/antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296 4.3.2. Gene therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 5. Applications of SPIONs for in vivo imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 5.1. Imaging modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 5.1.1. MR Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 5.1.2. Optical imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 5.1.3. Positron Emission Tomography (PET) Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 5.1.4. Conventional imaging compounds vs. MNPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 5.2. Dual imaging and drug delivery applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 1. Introduction Advances in nanotechnology and molecular biology are rapidly enabling the development of nanoparticles (NPs) with specific functional properties that address the shortcomings of traditional disease diagnostic and therapeutic agents [1���3]. Brighter, tissue- specific imaging probes are being developed with NP technology to visualize and help diagnose disease at its earliest stages, in some cases, even prior to disease manifestation [4,5]. Concurrently, NPs are being developed as drug carriers thanks to careful nanostructure construc- tion (tailored drug release characteristics, low immunogenicity, etc.) yielding improved treatment efficacy and reduction of unwanted side effects [6,7]. Significantly, these imaging and delivery facilities have been combined into unique NP formulations through clever combina- tions of nanoscaled materials, enabling simultaneous in vivo diagnos- tic imaging and drug delivery for real-time treatment tracking [7,8]. Among the broad spectrum of nanoscale materials being investi- gated for biomedical use, magnetic nanoparticles (MNPs) have gained significant attention due to their intrinsic magnetic properties, which enable tracking through the radiology cornerstone, magnetic reso- nance (MR) imaging [8]. This class of NPs include metallic, bimetallic, and superparamagnetic iron oxide nanoparticles (SPIONs) [8,9]. The latter of which has been widely favored because of its inoffensive toxicity profile [10���12] and reactive surface that can be readily modified with biocompatible coatings [13���16] as well as targeting, imaging, and therapeutic molecules [15���18]. This flexibility has led to SPION use in magnetic separation [19], biosensor [20,21], in vivo medical imaging [8,22,23], drug delivery [18,24], tissue repair [25], and hyperthermia [26] applications. Currently, a number of SPIONs are in early clinical trials or experimental study stages [8,9,15], and several formulations have been approved for clinical use for medical imaging and therapeutic applications. Notable examples include: Lumiren�� for bowel imaging [11], Feridex IV�� for liver and spleen imaging [27], Combidex�� for lymph node metastases imaging [28], and most recently, Ferumox- ytol�� for iron replacement therapy [29]. The physicochemical profiles of these SPIONs provide passive targeting, but not the higher level targeting offered by bioligands. Addition of bioactive molecules to the SPION surface can increase the targeting specificity of NPs [8,9,17,30,31], producing contrast agents that specifically illuminate targeted tissue and drug carriers that don't interact with healthy tissue [8,18,19,31���34]. Development in this area represents a majority of SPION research today. The creation of next generation SPIONs that can specifically target and eliminate or illuminate damaged tissue requires careful engineer- ing of the size, shape, coating, and surface modifications. Thorough consideration of each design parameter must be evaluated to produce a NP that can overcome biological barriers and carry out its function. In doing so, targeting molecules must be chosen based on their physical properties in addition to their binding characteristics, and integrated into the NP system in such a way that they remain functionally active. In vivo use of SPION imaging preparations require attention to each of these design parameters, while SPION drug delivery systems must additionally anticipate the routes of NP uptake by target cells and the controlled release of their payloads. Herein, we will review these design considerations and fabrication strategies for the development of NPs for in vivo imaging and targeted drug delivery. 2. Nanoparticle design considerations Before synthesis, MNP design requires fundamental understand- ings of the nature of the nanostructure as (1) a pharmaceutical construct that must navigate the body in search of its target, (2) a biocompatible entity that will not harm the patient, and (3) a contrast agent used in an external, biomedical imaging system. Here, we will consider the first of these areas, specifically looking at the physiolog- ical barriers that a MNP must overcome to gain access to its cellular target, and the NP's physical characteristics that can promote this functionality in vivo. 2.1. In vivo barriers Intrinsic to the body's defense system are a series of ���biological barriers��� that serve to protect the body against foreign entities, including injected therapeutics and contrast agents, keeping them from reaching their intended destinations [1]. These barriers can restrict NP function by blocking their movement, causing physical changes to them, or by inducing a negative host response using biochemical signaling [35]. Upon intravascular administration, NPs immediately encounter blood, a high ionic strength, heterogenous solution, that can induce NP agglomeration, altering their magnetic properties and inducing particle sequestration. Additionally, NPs can nonspecifically interact with plasma proteins (which can trigger the adaptive immune system), extracellular matrices, and non-targeted cell surfaces while in the blood stream [36]. In each case, the NP is in danger of prematurely binding to or being taken up by cells before reaching its target tissue. In addition to coping with the vascular environment, NPs must overcome various anatomical size restrictions which limit NP access 285 O. Veiseh et al. / Advanced Drug Delivery Reviews 62 (2010) 284���304

to target tissue (e.g. extravasation of lymph-targeting NPs from the blood vessels) [1]. These size limitations are especially stringent when targeting certain organs like the brain and kidney [37]. For instance, in the brain, endothelial cells and reinforcing astrocyte cells limit levels of pinocytosis and form tight junctions between cells at the blood- brain interface, yielding a structural and metabolic barrier referred to as the blood brain barrier (BBB) [38]. Here, only NPs of sufficient small sizes and appropriate physicochemical properties may pass the BBB. Biological barriers are not unique to extracellular spaces in fact intracellular barriers are a critical reason many drugs and drug delivery systems fail. NP systems are no exception. Once a cell-specific NP has bound to the membrane of its target, it is typically taken up by the cell through receptor-mediated endocytosis, where it is trafficked intracellularly via endosomal compartments for processing and destruction through acidification of the endosomes [39]. Most of these endosomes are then translocated into lysosomes where hydrolytic and enzymatic reactions completely metabolize macro- molecules. Many therapeutics, such as DNA and siRNA, are susceptible to lysosomal degradation, rendering them ineffective upon cellular processing. However, carriers can be engineered to avoid this fate by facilitating endosomal escape prior to lysosomal trafficking [35]. NPs that are able to demonstrate endosomal escape may still be required to breach additional biological barriers, such as the nuclear mem- brane, as is required for effective gene therapy. Each of these obstacles illustrates a demand placed on the engineers of a given system, and must be addressed in the preparation of the core and surface properties of the NP. 2.2. Physicochemical considerations NP pharmacokinetics and cellular uptake in vivo, including their ability to manage biological barriers, are largely related to NP physicochemical properties, including morphology, hydrodynamic size, charge, and other surface properties [40,41]. These properties are dictated by the types, structures, and orientations of the materials that comprise the NP. Typically, an MNP consists of a magnetically active core coated with a stabilizing shell to which targeting ligands and additional imaging modalities are anchored. Therapeutic agents can then be embedded in the shell structure or chemically bonded to its surface. At each stage of its design, the size, charge, hydrophobicity, shape, and orientation of the NP's constituent materials must be considered with regards to overall NP physiochemical properties. 2.2.1. Hydrodynamic Size NP biodistribution appears to be significantly influenced by its physicochemical properties [37,42]. Hydrodynamic size, for instance, (1) helps govern the NP concentration profile in the blood vessel [43��� 45], (2) affects the mechanism of NP clearance, and (3) dictates the permeability of NPs out of the vasculature [46]. In the case of the former, Decuzzi et al produced models suggesting that smaller sized, spherical NPs observed higher diffusion rates, increasing the NP concentration at the center of a blood vessel, thus limiting interactions with endothelial cells and prolonging the NP blood circulation time [45]. Hydrodynamic size also affects NP clearance from circulation [37,47���51]. For instance, it has been reported that small NPs (b20 nm) are excreted renally [47,52], while medium sized NPs (30���150 nm) have accumulated in the bone marrow [53], heart, kidney and stomach [52], and large NPs (150���300 nm) have been found in the liver and spleen [54]. While these size ranges provide general clearance mechanisms, other physical parameters simultaneously affect NP mobility. As previously discussed, nanoparticle size affects the ability of NPs to extravasate from the vasculature. While most endothelial barriers allow NPs b150 nm in diameter to pass, more stringent barriers, such as the BBB are far more restrictive. The BBB allows passive diffusion of only small (b500 Da MW), neutrally charged lipid soluble molecules, prohibiting N98% of all potential neurotherapeutics and contrast agents from passing through the BBB [55,56]. In addition, a vast majority of developed NPs have been unable to breach the BBB [38]. Consequently, this has become an area of intense research [38,56���59], with broad ramifications in the development of treatment strategies for brain tumors, Parkinson's, Alzheimer's, and Huntington's diseases [1,57,60���63]. In the quest to determine the influence of NP size on BBB permeability Sonavane et al recently reported that gold NPs of 15 to 50 nm in hydrodynamic size could permeate across the BBB, while larger NPs, specifically 100 and 200 nm sized could not [64]. However, it should be noted that reviews of the literature have suggested that BBB permeability is likely influenced by all physiochemical properties discussed here and NP size may not alone dictate NP permeability across the BBB [56]. 2.2.2. Shape In investigating the effects of NP shape on biodistribution, a limited number of comparative studies have been performed evaluating the biodistribution of non-spherical and rod shaped NPs [65���70]. It has been suggested that anisotropically shaped NPs can avoid bioelimina- tion better than spherical NPs [67]. In one notable study by Geng et al, the authors demonstrated a relationship by which an increase in the length-to-width aspect ratio of the nanostructure correlated with increased in vivo blood circulation time of nanostructures [70]. High aspect ratio shaped MNPs have also been evaluated in vivo and found to have similarly enhanced blood circulation times over the spherical counterparts [71,72]. Although these findings are promising more studies are needed to identify exactly what aspect ratios yield most dramatic influence on NP pharmacokinetics. 2.2.3. Surface properties NP charge and hydrophobicity can affect NP biodistribution by limiting or enhancing interactions of NPs with the adaptive immune system, plasma proteins, extracellular matrices and non-targeted cells [36]. Specifically, hydrophobic and charged NPs have short circulation times due to adsorption of plasma proteins (opsonization) which can lead to recognition by the reticuloendothelial system (RES), followed by removal from circulation [41]. Positively charged NPs can also bind with non-targeted cells (typically negatively charged) leading to non- specific internalization. In addition, hydrophobic groups on the surface of NPs induce the agglomeration of the NPs upon injection, leading to rapid removal by the RES. To limit NP-host interactions, surface engineering has led to the development of stealth NPs. Surface modification with molecules like the hydrophilic polyethylene glycol (PEG) have been shown to reduce the potential for opsonization through steric repulsion, prolonging NP circulation times [73]. The utility of organic coatings will be properly addressed in later sections. 2.3. Directing nanoparticles in vivo The specificity of NPs for select tissues is critical in both diagnostic imaging and drug-based therapies [16,74,75]. In both cases, nonspe- cific cell binding can place healthy tissue at risk. To limit non-specific binding, NPs have been engineered to have an affinity for target tissues through passive, active, and magnetic targeting approaches. Passive targeting uses the predetermined physicochemical prop- erties of a given NP to specifically migrate to a given tissue region. For example, targeting of solid tumor tissue can be achieved through passive mechanism termed enhanced permeation and retention (EPR) [76]. This phenomenon is based on the principle that tumor cells, in an effort to grow rapidly, stimulate production of new blood vessels (the neovasculature) that are poorly organized and have leaky fenestrations. This enables extravasation of small macromolecules and NPs out of the vasculature, into the tumor tissue [77,78]. Due to 286 O. Veiseh et al. / Advanced Drug Delivery Reviews 62 (2010) 284���304