Multifunctional nanosystems at the interface of physical and life sciences

Nano Today (2009) 4, 27—36

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nanotoday

REVIEW

Multifunctional nanosystems at the interface of physical and life sciences Won Hyuk Suh a, Yoo-Hun Suh b,∗, Galen D. Stucky a,∗ a

Department of Chemistry & Biochemistry, and Materials Department, University of California, Santa Barbara, CA, 93106, USA b National Creative Research Initiative Center for Alzheimer’s Dementia, and Neuroscience Research Institute, Medical Research Center, Department of Pharmacology, College of Medicine, Seoul National University, 28 Yeongeon-dong, Jongno-gu 110-799, South Korea Received 12 September 2008; received in revised form 14 October 2008; accepted 14 October 2008

KEYWORDS Nanoparticles; Nanomaterials; Nanostructures; Nanobiotechnology; Nanotechnology; Nano—bio interface; Biomedical engineering

Summary Multifunctional nanoparticle systems (MFNPSs) have seen a recent increase in the research effort put into the development of newer and improved systems around the world. This review covers the physical and biological aspects involved in nanoparticle systems having multiple functions such as optical and magnetic resonance imaging capabilities with incorporated bioactive molecules. Recent examples are covered based on a simple but logical categorization scheme. The promising platform of MFNPS in biomedical research is, in addition, discussed under the context of health (safety) and ethical concerns. © 2008 Elsevier Ltd. All rights reserved.

Introduction The chemistry and physics of materials change more notably with size at the sub-100 nm length scales. Transcription and translation within a cellular organism occurs within the sub-nanometer range, and with the advent of techniques and technologies available to create materials small enough to interact selectively with biological molecules such as nucleic acids and proteins, new opportunities to study cellular level activities are being made available. Latest advances in nanostructured material engineering

∗

Corresponding authors. E-mail addresses: [email protected] (Y.-H. Suh), [email protected] (G.D. Stucky).

geared towards biomedical applications have involved the fine tuning of particles that are micron in size and below (intravenously injectable [1]) to incorporate variety of components from fluorophores to magnetically susceptible domains. In this review, we will, first, summarize the internal and external structures of a mammalian cell in comparison to particle size and structure; and introduce important interactions that a multifunctional nanoparticle system (MFNPS) can have on biological systems present in or on the cell. Second, we will introduce the design concepts of MFNPS accompanied by excerpts of the latest state-ofthe-art particle systems. Third, several realistic concerns of biomedical research utilizing MFNPS will be discussed with some key examples such as the blood clotting effects directed by inorganics. Some new directions are described on how to interface and develop physical and life science

1748-0132/$ — see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.nantod.2008.10.013

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Figure 1 A cell as a multifunctional entity. (A) The breakdown of the cellular machinery shows that a mammalian cell is essentially a multifunctional particle comprised of internal and external components. The external components are (1) the cell membrane, (2) surface proteins/ion channels, and (3) the cytoskeleton, while the internal components are (4) the cytosolic organelles (i.e. ribosome, endoplasmic reticulum, golgi apparatus, mitochondrion, vacuole, lysosome and centriole), and (5) the nucleus (which contain nucleic acids). Another major component in a living cell consists of the active biological processes that are on-going in a constant and dynamic manner. This involves proteins, metal ions, nucleic acids, amino acids, external stimuli and other biomolecules. (B) Multiple cell types exist differing in their function, shape and size. (C) The cell membrane consists of many functional biomolecules which include multiple forms of functional proteins, displayed carbohydrates, and the lipid bilayer. A sub-10 nm nanoparticle can directly interact with such biomolecules present on or inside a cell as shown in the illustration.

efforts together which can lead to a successful end product. Lastly, we will additionally talk about safety (nanotoxicology) and ethical issues surrounding nanoparticle-based research.

Nano—bio interface and the cell as a functional particle The cellular machinery is comprised of five major components: (1) the cell membrane, (2) receptors/proteins/ metabolites, (3) the cytoskeleton, (4) the cytosolic compartments (simply cytosol), and (5) the cell nucleus (Fig. 1). Nanotechnology utilized in biologically oriented applications have increased in the past several years which is often termed as nanobiotechnology or bionanotechnology. Proteneous molecules and nucleic acids (biomolecules) have been the focus in many instances [2—6]. Sub-micron particles are small enough to interfere or alter cellular level functions so that their interactions with biomolecules play a critical role (Fig. 2). In the case of particle-based research, multiple research efforts involving the use of inorganic, organic, and composite (hybrid) nanoparticles have been conducted to synthesize, characterize and then utilize them in biological systems for drug delivery and cellular level bioimaging [7—11]. In recent years, an increasing number of papers examine the interactions between a protein and a nanoparticle [12—15]. As shown in Fig. 2, engulfed 100 nm nanoparticles (right side of Fig. 2C) and a 1 ␮m porous microsphere (left side of Fig. 2C) are comparably much smaller than a mammalian cell (top left panel, Fig. 2A). Practically, nanoparticles or nanostructured materials interfaced to living systems (e.g. cells to animals) will encounter numerous associative and/or dissociative interactions in the sub-micron range with the surrounding host biological system which include nucleic acids, proteins, ions, and water. Surface chemistry (charge and functionality) is a key feature that will decide many of these interactions.

In fact, blood clotting rates and hemolytic activity are highly dependent upon metal oxide surface environments (e.g. morphology, charge, porosity) as well as material composition [16—18]. Such results suggest that the choice of material and fine tuning of surface physicochemical properties to match the space and temporal domains of a biological system is essential. Surface charge differences, from the biology side, in stem cells and their differentiated states allowed the separation of different cell populations using dielectrophoresis in a microfluidics device which was a result of gene expression level differences governing surface protein production at different stages of stem cell differentiation [19]. Cell separation, in general, is achieved by identification of surface markers, precisely attaching optical probes to specific targets, and by physical sorting based on optical detection methods in an automated mechanical device such as a FACS (fluorescence assisted cell sorting) machine. The cell surface-displayed makers commonly used for human embryonic stem cells, for instance, are SSEA-3, -4, TRA1-81, TRA-1-60 while other markers exist for neural stem and precursor cells, and differentiated neurons [20—22], etc. A suspension of cells undergoing population sorting (via FACS) can be further analyzed using microarrays as illustrated in Fig. 3A which shows how undifferentiated stem cells’ gene expression profile is different from that of embryonic bodies [21]. This can be considered as an equivalent of a size distribution graph (Fig. 3B) of particles from the physical sciences which is commonly analyzed via electron microscopy (as shown) and/or dynamic light scattering. Cells are distinguishable based on surface characteristics (i.e. antigen—antibody interaction and surface charge) as well as gene expression profiles so that combining such efforts with surface research efforts from the physical sciences can be expected to lead to a wide range of cellular responses and activities. One thing to note, however, with regards to surface charges (i.e. isoelectric points) is that such characteristics dramatically change depending upon the sur-

Multifunctional nanosystems

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Figure 2 Cell vs. 100 nm particle vs. 1 ␮m particle. (A) SEM image of mammalian cells and micron sized particles. (B) Engulfed particles partially exposed on a thin film substrate. (C) Magnified SEM image of black box in (B). One micron porous particle (left) and 100 nm multifunctional particles (right) are seen wrapped under a cell membrane. The particles were treated for 3 days in a microglia cell culture. (D) Fluorescence microscopy image of cells that engulfed fluorescent sub-micron metal oxide particles. The yellow arrow shows an internalized particle aggregate that does not appear to have altered the particular cell’s ability to under go mitosis, a key cell function involving the genetic code. The blue color represents DAPI staining of the cell nucleus and the red color represents fluorescent particles emitting in the red. SEM images were taken at the California NanoSystems Institute (CNSI), now Elings Hall, UCSB using an FEI XL40 SEM.

rounding solution chemistry and physics (e.g. osmolarity, ionic strength, nanoparticle solubility, metal ion valency) [23,24] so that not only is it important at the synthetic stages but it will be also very important at the application stages where nanoparticles (or any other materials) are interfaced to biological systems creating the nano—bio interface. The three charge-based excerpts described above show that precisely understanding both materials’ surface charges as well as the biological entities’ surface chemistries or changes thereof will be vital in properly investigating biological systems undergoing constant dynamic processes. Efforts studying the nano—bio

interface due to such characteristics now-a-days should involve properly understanding both the physical and biological sides of research leading up to a multidisciplinary research initiative. Focused in vitro research dedicated towards understanding the changes in cell biology (i.e. transcription, translation, viability, necrosis, apoptosis, and endo/exocytosis) interfaced to nanostructures should be studied within the context of the relevant cell biology. Research involving MFNPS should utilize such information in the initial stages of particle system development and in concert with multiple biological experiments to analyze DNA, RNA, and proteins to enable an increased level of

Figure 3 Population analysis of cells and particles. (A) Gene expression profile of embryoid bodies vs. that of undifferentiated pluripotent stem cells (PSCs). Adapted from Ref. [21] with permission from Elsevier. (B) Size distribution profile of sub-200 nm SiO2 nanoparticles (subset) with the representative TEM.

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Figure 4 Generalized synthetic scheme of a multifunctional nanoparticle system (MFNPS). Metal alkoxides (i.e. Si(OR)4 , Ti(OR)4 where R = H, hydrocarbon) undergo rapid to slow hydrolysis and condensation reaction steps under acidic or basic conditions to form a metal oxide network (matrix). Depending on the presence and nature of additives, MFNPS characteristics will be precisely determined. Matrix density and the degree of polymerization are highly dependent upon reaction conditions that can be the type of additive, time of aging, reaction temperature, and purity of additive contents. Four key components are (1) an optical probe (e.g. fluorophore), (2) a secondary imaging probe (e.g. magnetically susceptible or radio-active), (3) porous structure and/or chemical functionality, and (4) biologically active load or cargo (i.e. antibodies for targeted delivery; drugs, nucleic acids and other proteins for therapeutic applications or control over biological processes).

understanding that nanostructures will have on the cellular machinery (Fig. 1). It should also be noted that multitudes of cell types exist in higher organisms such as animals and humans, which means that our body is, in fact, a compilation of multifunctional nanosystems. This is why the process of analyzing biological functions within an individual cell and cells interfaced to other cells, connective tissues, and other materials also must be treated by a systems analysis procedure. In a similar manner, constructing MFNPS from sub-components is also challenging but, at the same time, rewarding.

MFNPS (multifunctional nanoparticle system) Key components of MFNPSs (Fig. 4) are four- to five-fold depending on the particle system’s internal and external physicochemical properties: (1) a matrix component provides the basis for additional component inclusion (usually

ten to hundreds of nanometer in size), (2) a fluorescent optical probe (e.g. Rhodamine, FITC) for optical microscopy, (3) a magnetically susceptible sub-domain (e.g. Fe3 O4 ) for magnetic resonance (MR) imaging, and (4) pores or functionalities that allows the incorporation of small molecules (i.e. drugs) or biomolecules (i.e. proteins and nucleic acids). Multifunctional nanoparticles can be comprised of mainly organic components or inorganic components or somewhere in between as a hybrid nanocomposite. ISI Web of Science search (nanoparticle* AND multifunction*) yields over 500 hits for MFNPS (accessed 11 October 1008). Thus, distinguishing MFNPSs based on materials’ chemical components (aspects) may not be accurate (or in depth) enough to fully help end-users (in the biomedical community) to effectively understand what they are capable of and how best to utilize them. In this review, we have categorized MFNPSs based on their internal and external component and functional analyses (Fig. 5).

Figure 5 Categorization of multifunctional nanoparticle system (MFNPS). MFNPSs can be distinctively categorized into four (or five) distinctive particle types based on, mainly, sub-domain compositions and overall size. Type I MFNPSs are non-porous spherical inorganic based on sub-200 nm nanoparticles with two or more components. Type II MFNPSs are sub-200 nm spherical nanoparticles that are either porous or can incorporate bioactive components such as antibodies and drug molecules. Type III MFNPSs are sub20 nm nanoparticles with bioactive functionality on the surface (as well as having sub-components) usually installed after phase or ligand exchanging. Lastly, type IV MFNPSs are non-spherical nanoparticle systems (having high-aspect ratio morphologies) that have multiple sub-components such as fluorescent tags, imaging probes, drugs and antibodies.

Multifunctional nanosystems

MFNPS type I: sub-200 nm and non-porous nanoparticles Type I MFNPSs are sub-200 nm spherical non-porous metal oxide (i.e. silicon dioxide) based systems which contain two or more physicochemically active sub-components. Inorganic particle-based systems are highly tunable so that size and internal/external content modification can be easily achieved and generally involve sol—gel synthesis methods as described in Fig. 4. In 2005, Santra and co-workers reported a type I MFNPS that incorporated Gd3+ /ligand (MR imaging) and [Ru(bpy)3 ]2+ (fluorophore) that was terminated with an amine group available for bioconjugation. This system was MR and fluorescence active but also was radio-opaque. In subsequent years, Rieter et al. [25] and Kim et al. [26] have reported sub-50 nm SiO2 based MFNPS for cancer-specific optical and magnetic resonance (MR) imaging probes in mice. Incorporated MR agent was Gd3+ /ligand and the fluorophore was [Ru(bpy)3 ]2+ . One thing to note about this inorganic compound is that it is electrochemically active, which can offer additional analysis parameters [27—31]. The Lin group also combined layer-by-layer methods with conventional sol—gel methods to incorporate not only the MR and fluorescent probes but also K7 RGD peptides [26] which can target cancer cells which was a peptide utilized earlier by Suslick and Boppart [32]. Lee et al. reported an MFNPS that incorporates Fe3 O4 and fluorescence probes that have multiple functional groups such as amine, thiol, and hydroxy [33]. Shin, Cheon and coworkers reported a 30—40 nm MFNPS which was a result of fusing together amine-functionalized dye-doped SiO2 sub30 nm nanoparticles and thiol-functionalized Fe3 O4 using sulfo-SMCC cross-linker. The MFNPS was successfully tested for its labeling and imaging capabilities on HEK293T (kidney) and CHP-134 (neuroblastoma) cells [33].

MFNPS type II: 50—200 nm (or larger particles) Type II MFNPSs are approximately 50—200 nm spherical nanoparticles that contain porous or bioactive (i.e. drugs and antibodies) compartments that allow molecular species incorporation or specific targeting of cellular level systems. Additional sub-components include one or more probes for imaging related applications and, traditionally, micronsized examples have been investigated more. Haam and co-workers reported 70—90 nm sized PLGA (poly(lacticco-glycolic acid) copolymer spheres incorporating Fe3 O4 , doxorubicin (DOX), and the Herceptin (HER) antibody which has been used to successfully target breast cancer cells [34]. In contrast to inorganic-based particle systems, biodegradable or biocompatible polymers such as poly(L-lactic acid) (PLLA), bovine serum albumin (BSA), and PLGA have been developed some time ago and is widely used for the controlled delivery of drugs and proteins in spherical forms [35—42]. Huperzine A (an Alzheimer’s disease drug candidate) was encapsulated in PLGA and are used to treat memory-impaired animals [43,44]. Such polymeric systems can be further modified to include sub-domains that can offer imaging and targeting capabilities commonly associated with inorganic MFNPSs. From the inorganic side, Hyeon and co-workers created <200 nm SiO2 hollow and

31 mesoporous spheres that incorporates Fe3 O4 , CdSe/Zn nanoparticles, and a hydrophobic NSAID (non-steroidal antiinflammatory drug). In particular, ibuprofen was shown to be released in vitro in a continuous fashion over a period of time while the MFNPS was responsive to magnetic forces and fluorescent when excited using a light source. The same group more recently reported an iron oxide@mesoporous SiO2 core—shell system functionalized with dyes and/or PEG that was used to, first, label MCF-7 cells, second, study in vivo T2 -weighted MRI and fluorescent imaging in animal studies, and, third, DOX drug loading and releasing experiments on SK-BR-3 cells [45]. In a different study by Mou, Huang and colleagues, silica encapsulated Fe3 O4 core—shell nanoparticles were fused with fluorescent (FITC) and mesoporous moieties side-byside which were shown to be non-cytotoxic (shown via MTT assay) to work with NIH 3T3 [46,47], bone marrow stromal cells (rMSCs) [47], and human mesenchymal stem cells (hMSCs) [48]. The same group of researchers also concluded that uptake of the as-synthesized fluorescent and mesoporous SiO2 nanoparticles (100 nm in size) are dependent upon surface charge and cell population matching [49]. Zink, Tamanoi and co-workers in a similar manner created mesoporous and fluorescent nanoparticles with reduced agglomerating characteristics (via surface chemistry modulation) and demonstrated anti-cancer drug delivery in cell cultures [50]. The same group of researchers, not long ago, reported a similar type of mesoporous particle system but with fluorescent, magnetic, and therapeutic subcomponents all embedded into one overall structure [51]. Stucky, Wang and co-workers also developed a multifunctional and porous inorganic system with an overall size of approximately 1 ␮m and below using an ultrasonically [52—58] or a nozzle [59—61] generated aerosol synthesis technique to produce fluorescent microspheres with high surface area which creates highly disaggregated spheres [62—64]. Such aerosol processes are used, also, for spray drying therapeutics and biodegradable polymers that can incorporate sub-domains such as fluorescent quantum dots and drug molecules [1,65,66].

MFNPS type III: sub-20 nm nanoparticles Type III MFNPS consists of sub-20 nm nanoparticles with organic ligands or biomolecules stabilized (passivated) onto the nanoparticle surface and are, in most cases, first synthesized in the organic phase and then stabilized (via phase exchange) into an aqueous media. A team of researchers headed by Ying of IBN (Institute of Bioengineering and Nanotechnology) in Singapore, synthesized silica-coated semiconductor CdSe-based quantum dots (fluorescent) that were grown over Fe2 O3 nanoparticles (magnetic core) via microemulsion methods and demonstrated that magnetic and luminescence properties could be controlled by varying the silica surface chemistry by silanization. The MFNPS synthesized in this study was functionalized with oleic acid conjugated PEG (poly(ethylene glycol)) molecules to label the cell membranes of HepG2, NIH-3T3, and 4T1 cells [67]. In a similar fashion, Santra and co-workers prepared SiO2 -based sub-20 nm MFNPS which encapsulates CdS:Mn@ZnS fluorescent nanoparticles in the presence of MR active Gd3+ and

32 chelating ligands. The surface of these nanoparticles were conjugated with TAT peptides and then utilized for rat brain labeling studies [68,69]. Magnetic nanoparticles 10—20 nm in size (e.g. nickel-based magnetic core with oxide shell) with surface functionality allows the magnetic separation of proteins with histidine tags from cell lysates in the presence of Ni2+ [70,71]. Cheon and colleagues prepared a dumbbellshaped bifunctional nanoparticle functionalized with bioactive molecules such as antibodies; Au NP served as an optical probe and FePt NP served as an MRI probe [72]. Similarly, Sun and co-workers prepared Au-Fe3 O4 nanoparticles functionalized with PEG conjugated antibody from the oxide side and amine/thiol terminating PEG from the gold side to conduct biomedical research using A431 cells via optical and MR imaging [73]. Glioma cells were targeted and imaged optically and via MRI by Zhang, Olson and co-workers. The MFNPS utilized by this team of scientists were 9—12 nm iron oxide nanoparticles functionalized with Cy5.5 (near-IR fluorescent probe), Chlorotoxin (glioma targeting bioactive molecule), and PEG (biocompatible stealth coating) [74,75]. On the (small interfering RNA) front, Bhatia and coworkers utilized a commercial quantum dot (QD) that was PEGylated and functionalized with a tumor-homing peptides along with the nucleic acid. The target gene was successfully down regulated with specificity of the cell and nucleic acid [76]. In a similar manner but using Au nanoparticles, Mirkin and co-workers decorated an Au NP (approximately 13 nm) with thiol functionalized oligonucleotides and successfully showed that specific translational process can be shut down in a non-toxic manner [77]. Nie, Gao and colleagues, more recently, constructed a unique MFNPS incorporating siRNAs, QD (optical and electron microscopy probe), and a protonsponge which showed over 10-fold increase in RNAi (RNA interference) efficiencies and an over 5-fold decrease in overall cytotoxicity in MDA-MB-231 cells by particle life time management [78].

Non-spherical MFNPS: type IV Type IV MFNPS is a non-spherical system with high aspect ratios (i.e. rods and tubes) that have multiple components including imaging probes and targeting molecules (i.e. peptides and antibodies). Type IV MFNPSs are anticipated to have a different biological fate in living systems from cells to whole bodies, which arises from differences in surface physicochemical properties, fluid dynamics, etc. Discher and colleagues, in a recent study showed that particle flow and subsequent delivery of drugs are affected by shape of the nano-sized material in vivo [79]. Non-spherical shapes (filaments) increased particle residence times by 10-fold compared to its spherical counter-part. This characteristic naturally prolonged drug release time and also resulted in cell uptake efficiency changes. A Ni/Au hybrid nanorod (100 nm diameter with two 100 nm segments) was decorated from the Ni side with cationic linkers to facilitate the binding of negatively charged bioactive molecules such as plasmids while thiol terminated fluorescent tags were put on from the Au side [80]. This metallic species-based MFNPS was utilized for gene delivery research in mammalian systems both in vitro and in vivo. An oxide nanotube-based MFNPS (50 nm diameter, 200—500 nm length) was developed by Lee

W.H. Suh et al. and co-workers for biosensors and to achieve controlled release of small molecules [81,82]. The SiO2 nanotubes were prepared from electrochemically synthesized porous membranes and then decorated with iron oxide nanoparticles as the MR agent with internally and externally functionalized antibodies and an internally loaded drug cargo. Further studies showed that the silica nanotubes are reasonably non-cytotoxic (at lower concentrations of <0.05 ␮g/mL) and can be utilized in multitudes of cell labeling experiments [83—85]. Low working concentrations compared to other nanostructured inorganics (e.g. 1 ␮m sized porous TiO2 particles, although spherical, can be used at 100- to 1000-fold concentrations [53]) warrants more extensive biological experiments on such non-spherical system in order to gain key insights into nanoparticle morphology-based cell biology control. A different templating chemical-based approach was used by Lin and co-worker to prepare a high aspect ratio MFNPS. Metal-organic framework-based nanorods were first synthesized, stabilized and then a shell of SiO2 was formed around each particle that eventually had a diameter of 40 nm and a length of 100—125 nm. Based on pH, the MOF moiety can dissociate and liberate the constituents in a controlled manner leaving behind a hollow nanorod [86]. On the other hand, lanthanide metal ions (i.e. Eu3+ , Gd3+ , and Tb3+ ) doped into the MOFs make MR imaging and fluorescence imaging viable [87].

MFNPS: a short summary and the reality Multiple examples of different types of MFNPSs are described above and it is apparent that multitudes of systems have been designed for incorporated small moleculebased therapeutics [25,33,46,69,87—94]. Figs. 4 and 5 illustrate the overall theme for MFNPS research in a simplified manner. Successfully engineering and achieving a balance between practicality (i.e. reasonable synthesis yield, good synthetic reproducibility, and simple enough usage protocol for the biological community) and design complexity (i.e. spherical vs. non-spherical, porous vs. nonporous, overall size) of MFNPS will be two major components to consider to successfully translate a materials science and engineering product into a useful biomedical application. Three realistic issues to consider, however, for biomedical applications of MFNPSs are, first, the aggregation of nanoparticles in aqueous solutions containing more than just water molecules, second, dissolution (biodegradation or nanoparticle degradation) properties, and, third, the blood clotting properties of particulate matter in vivo. Methylphosphonate functionalization [50,51], peptide conjugation [68,95] and PEG polymer coatings [45,96] onto nanoparticles have been shown to be effective on their disaggregation in aqueous media by basically modulating the surface properties of the nanoparticles in order to reduce their accessibility by the immunological mechanisms [97,98] present in cells and higher organisms. The majority of the MFNPSs covered in this paper are inorganic based with only a handful of examples coming from the organic side (i.e. degradable polymeric matrix particles). The silica platform is quite attractive in the sense that the base sol—gel chemistry (i.e. Stöber synthesis [99]) is well understood, which allows synthetic materials scientists to have precise

Multifunctional nanosystems control over the MFNPS contents [100]. In vivo distribution of hydrophilic silica nanoparticles, for instance, in rats show that the silica levels of accumulation in organs depend predominantly on size; the lung, heart, and spleen biodistribution is lower than 1% over a 2-day period for sub-250 nm SiO2 while the liver, kidney, skin, bone, gastrointestinal tract shows signs of retention starting as high as 20% but reaching sub-5% levels in 48 h [1]. The same study also compared bioavailability (24 h) of injected nanoparticles in the animal, which showed approximately 40% still being present in the liver and blood. Sanchez and co-workers showed that mesoporous SiO2 dissolved in cell media conditions while TiO2 does not [101] suggesting possible short-term and longterm uses of porous materials such as SBA-15 [102,103], MCF [104,105], and porous TiO2 [53,62—64,106,107] for biomedical applications. Although such findings may provide initial motivations to further develop the use of inorganic-based MFNPSs for biomedical applications, the results obtained to date do warrant, however, more extensive in vitro and in vivo biological investigations. Nanotechnology Characterization Laboratory (NCI) recently published protocols for precisely analyzing the possible role of nanomaterials in hemolysis (blood cell damage) [108]. Interestingly, Stöber and co-workers (who linked aerosol particulates to inhalation toxicology [109]) conducted similar research but only with materials available mostly in nature [18,110] some 40 years ago and much has happened since then in terms of the materials that require further testing using such bio-characterization methods. Stucky and co-workers have successfully demonstrated that surface physicochemical properties, ionic strength and nanostructures can promote blood coagulation which has been a key research initiative on external wound healing [16,17,60,61,111,112]. The blood clotting and hemolytic properties of particulate matter [113—116], however, inside a living system potentially can be a problem that needs to be further addressed for MFNPSs to go past animal studies and into clinical stages.

MFNPS: biomedical research and nanotoxicology Material scientists’ increased understanding of the time domain and the internal and external structures of living cells (Fig. 1and 2) that can be potential targets will be a key factor in the further development of MFNPS-based nanobiotechnology. At the same time an increased effort from the life sciences community to recognize the potentials of such tools will, in addition, be an important step forward for bionanotechnology utilizing MFNPS. Tailoring the size, components, and surface properties via physicochemical methods and nanotechnology will allow research efforts to develop well-characterized sub-micron MFNPSs for biomedical research, that includes cancer research, wound healing, the treatment and diagnosis of nervous system and brain related abnormalities, and regenerative medicine involving neural and stem cell engineering. Multiple review articles have been written recently with a focus on ‘size’ being a key issue in drug delivery to the brain past the blood—brain barrier [117—120]. Such a factor, however, will be a paramount issue where overall structures of nanomaterials have a dynamic range. A major question that must be

33 answered is when high aspect ratio materials (e.g. carbon, silica, and titania nanotubes) are involved, how different functional groups on the surface will affect in vitro and in vivo activities [79]. Biomedical research involving the use of MFNPS to achieve solutions for multiple biological problems will benefit more if lessons learned from nanotoxicology research efforts [121—124] are more precisely defined and investigated. Hurt, Kane and colleagues, for example, showed that adsorption of small molecules and nutrients onto carbon nanotubes due to their sufficient surface area may be significant enough to affect cell biology in cultures [125]. In another example shown by Krug and co-workers, several different cell viability (cytotoxicity) assays (i.e. MTT, WST-1, INT, and XTT) were tested on single-walled carbon nanotubes (SWCNTs) which turned out to affect one of the assays while not essentially affecting the others [126]. Such findings demonstrated why researchers assessing nanotoxicology must use multiple assay systems and develop tailored and standardized protocols for testing any synthetic nanomaterial. In fact, a group of scientists headed by Schreiber and Weissleder recently investigated toxicological effects of some standard nanomaterials via high throughput screening methods [127]. More recently, Nel and co-workers showed that a highly systematic comparison of TiO2 , ZnO and CeO2 nanoparticles based on precisely defined physicochemical (i.e. size, crystal structure, size, surface charge, and solubility) and biological (i.e. membrane potential, oxidative stress, uptake route, viability, immunological response, and immunocytochemistry) properties can effectively distinguish potential toxic materials from non-toxic ones [128]. UCSB, in Fall 2008, announced a new EH&S (Environmental, Health & Safety) safety fact sheet on the safe usage and handling of engineered nanomaterials and this guideline aims at the proper administration of safety training related to the class of materials and increasing the awareness of toxic potentials of nanomaterials in general [129]. Safety concerns with nanostructured materials have not yet resulted in a state or national wide regulatory policy in the US (with the exception of the city of Berkeley, CA [130]). It should be pointed out that multitudes of MFNPS research efforts should be self-regulated by the researchers, not only as related to health risks but also the way research is conducted with animals and cellular organisms where experimental planning and waste management can potentially be a complex and delicate process. Biological systems are being doped with engineering nanostructured materials, which are still a very much unknown entity when it comes to environmental and medical impact. The most viable option, at the current stage, is for individual research groups and multidisciplinary teams working at the nano—bio interface to establish a close relationship with their host institution’s laboratory safety and biosafety organizations and IACUC (Institutional Animal Care and Use Committee) or other safety/policy committees members in order to properly define and deal with potential ethical and health related agendas.

The future of MFNPS Nanotechnology is now at a stage where the applications are being well sorted out; catalysis, energy research, electron-

34 ics, and biomedicine are the prime candidates to benefit the most out of nanoscience. Nanobiotechnology in particular will see increased research efforts with particle-based technology. Improved particle design (incorporating both inorganic and organic), synthesis of MFNPS and final usage as therapeutic or diagnostic agents for biomedical research will aid research initiatives such as regenerative medicine involving neural and stem cell engineering to push forward and, in time, translate into clinical stages [131—134]. The blood clotting research efforts, as mentioned above, have resulted in commercial products that can be utilized for civilian traumatic injuries in accidents or when military personnel are deployed in war zones and blood loss means potential loss of life [135,136]. Such results came about when the role of inorganics controlling the bioprocesses involved in the blood coagulation cascade was properly understood. We envision that similar research insights and efforts can offer us new innovative ideas on the research front of MFNPSs and result in others needed applications in the biomedical field.

Acknowledgments All illustrations were designed and created by Dr. Won Hyuk Suh (WHS). SEM (Fig. 2) and TEM (Fig. 3) were taken at the UCSB California NanoSystems Institute (CNSI) using an FEI XL40 SEM and an FEI T20 TEM. The fluorescence microscopy image in Fig. 2 was taken at the Microscopy Suite at the UCSB Bio II building using an Olympus DSU microscope. Financial support from National Creative Research Initiative (CRI) Support from BK 21 Human Life Sciences (Korea), USA Office of Naval Research (N00014-06-1-0145), USA National Science Foundation (DMR 02-33728) is gratefully acknowledged. WHS thanks the Otis Williams Postdoctoral Fellowship in Bioengineering, UCSB (via Santa Barbara Fund) and the Drickamer Predoctoral Research Fellowship, UIUC for past, present, and future supports. Special thanks to Professors Kenneth Suslick, Patricia Holden, Kenneth Kosik, and Matt Tirrell for increasing the authors’ understandings of the multiple nano—bio interfacial problems. In addition, WHS would like to thank the NIH sponsored CHOC/Burnham (now Scripps) human embryonic stem cell training course and its directors, Dr. Philip Schwartz and Prof. Jean Loring, and the staff members. Lastly, we thank Dr. Schwartz on providing the original source file of Fig. 3A.

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sound based synthesis tools. Subsequently, he moved to the University of California, Santa Barbara for his postdoctoral work in materials and bioengineering where he is working on porous and nanostructured hierarchical materials for biomedical applications which include stem cell engineering and nanotoxicology under the guidance of Profs. Galen D. Stucky, Kenneth Kosik, Matthew Tirrell and Patricia Holden. He was the recipient of the Harry G. Drickamer Pre-doctoral Fellowship, UIUC (2005—2006) and is the current recipient of the Otis Williams Postdoctoral Fellowship in Bioengineering, UCSB (2008—2010). Yoo-Hun Suh received his M.D. (1973) and Ph.D. (Pharmacology, 1981) from the College of Medicine, Seoul National University, Korea. Subsequently, he joined the Dept. of Pharmacology and is now the Chairman of his department. He serves as Directors for the National Creative Research Initiative Center for Alzheimer Dementia (AD), Neurosci. Res. Inst. (SNU), and Cognitive Sci. Inst. (SNU). His research interests are molecular biology, biochemistry, and pharmacology of AD with a recent focus on stem cells and nanobiotechnology. He has served as Presidents to six different societies and centers and was the Medical College Dean at Kangwon National Univ. (1997—1999). He received many awards which include the 5.16 National Prize (2004), Eui Dang Medical Award (2004), National Govern. Medal (Woong Bie Medal) (2002), and the Yu Han Grand Prize (2002). He serves on the editorial boards of J. Neurochem., J. Mol. Neurosci., J. Neurosci. Res., and Neurochem. Res. Galen D. Stucky earned doctorate from Iowa State University in 1962. He held positions at the University of Illinois at UrbanaChampaign, Sandia National Laboratory, and DuPont Central Research and Development before joining the faculty of the University of California, Santa Barbara, in 1985, where he is Professor in the Department of Chemistry & Biochemistry and the Materials Department and a member of the Interdepartmental Program in Biomolecular Science and Engineering. His current research interests include molecular assembly of nanoscale to macroscale components of composite systems; the interface of inorganics with biomolecules; chemistry associated with the efficient utilization of energy resources; and understanding Nature’s routes to organic/inorganic bioassembly. He has published over 600 scientific articles and has been awarded 13 patents. Recent honors include the ACS Award in Chemistry of Materials (2002), the IMMA (International Mesostructured Materials Association) Award (2004) and the Department of Defense’s Advanced Technology Applications for Combat Casualty Care (ATACCC) Award (2008). He was elected Fellow, American Academy of Arts and Sciences, in 2005.