Multifunctional magnetic nanoparticles1 are particles about one hundred-thousandth of the diameter of a human hair that combine biologically active molecules with metal particles (occasionally, metal compounds) that respond to a magnetic field. They are considered multifunctional because of the variety of their potential applications, including, in biomedicine, capturing bacteria and purifying proteins. Multifunctional magnetic nanoparticles are recent examples of bionanomaterials. Fabrication and use of such materials is known as nanobiotechnology, and is the branch of nanotechnology that focuses on biological and biochemical applications.2 Nanobiotechnology deals with the many molecular processes that occur within living cells on very small (nano-) scales. Improving knowledge of such processes is providing an understanding of many complex cellular functions.
The commonality of “nano” and “bio”
While the objective of nanoscience (nano) is to explore objects and systems that have sizes between 1 and 100 nanometers (nm) in at least in one dimension (Figure 1), biological science (bioscience or bio) primarily focuses on the components and functions of cells. Although the sizes of whole cells range from thousands to tens of thousands of nm, the components of cells usually have sizes below 100 nm in at least one dimension. For example, cell membranes are less than 10 nm thick; the cytoskeletons in mammalian cells are nanofibers of actins and microtubulins tens of nm in diameter; and double-stranded DNA is a nanowire just a few nanometers wide. Therefore, nano and bio share the same range of sizes (1-100 nm), which has allowed scientists to explore the interactions between the two.
Figure 1. Comparison of the sizes of atoms, nanoparticles, and biological entities.
Most importantly, the sizes shared by nano and bio are in a special range. Within this range, new phenomena emerge—that is, the behavior of a group of objects differs drastically from that of each individual object in the group. Figure 2 shows examples of objects and structures in nanoscience or biological science. Figure 2A shows a memory cell from a contemporary computer memory chip; made of a few thousand silicon atoms, it can store bits of information, yet a single atom of silicon (one tenth of a nanometer in diameter) cannot. A phospholipid molecule is useless as an individual molecule; however, when millions of the phospholipids self-assemble in water, they form a membrane 3-9 nm across (Figure 2B) that compartmentalizes a myriad chemical reactions and may have led to the emergence of life. In Figure 2C, although filaments of actin molecules control cell movement, an individual actin molecule has little power to modulate cell behavior. In Figure 2D, although bulk iron-platinum alloy is a “hard” magnetic material (meaning that a large magnetic field is needed to change its magnetization), magnetic nanoparticles of the alloy less than 2 nm in diameter are said to be superparamagnetic—their magnetization is easily influenced by a magnetic field. Clearly, these examples indicate that nanostructures are unique and differ from both the bulk materials and the individual molecules or atoms. Indeed, life itself is fundamentally a collection of processes taking place at the nanoscale within cells.3 It is important, therefore, to understand the interactions between nanomaterials and biological systems and to explore their applications.
Figure 2. Electron-microscope images of some objects in nanoscience or biological systems. (A) A 32 nm node for flash memory4 ; (B) the membrane (white arrow) of a bacterium5 ; (C) filaments of actin (a cytoskeleton component) 6 (©2008, American Chemical Society) ; (D) Iron-platinum magnetic nanoparticles (approximate size 4 nm) (©2003, American Chemical Society).
Molecular interactions as the foundation of nanobiotechnology
Although the functional basic unit of life is the cell, all the processes occurring inside and among cells are sophisticated molecular interactions. Advances in the field of molecular biology continue to reveal details of cellular activities at the molecular level. These have resulted in many extraordinary successes, including:
- the discovery of the structures and functions of DNA and RNA;
- the elucidations of the structures and functions of numerous enzymes; and
- advancements in molecular medicine.
Because molecular interactions dictate biological processes, it would be impossible to explore and apply nanomaterials in biological science without a fundamental understanding of the molecular interactions between nanomaterials and biological entities.
Design and fabrication of multifunctional magnetic nanoparticles
Because of the unique response of nanoparticles to a magnetic field, simply attaching them to biofunctional molecules leads to multifunctional nanostructures. Magnetic nanoparticles, therefore, are a favorite choice for the development of multifunctional bionanomaterials. To construct multifunctional nanoparticles, it is necessary to consider both the size and surface chemistry of the nanoparticles in order to control the molecular interactions between nano and bio.
Choice of sizes. Two simple criteria help define the optimal size of magnetic nanoparticles to achieve high affinity, selectivity, and sensitivity in interactions with biological objects:
(1) the nanoparticles should be large enough to allow the presence of multiple ligands (substances, such as neurotransmitters, that form complexes with biomolecules by binding to specific sites). This will allow multiple interactions with a nanoparticle and allow it to bind tightly with biological targets.
(2) the nanoparticles should be small enough to have high surface-to-volume ratios, to be stable when dispersed in a liquid as a colloid, and to move quickly (thus allowing fast reactions). For example, to separate biomacromolecules such as proteins, the size of the nanoparticles should be comparable to the size of the biomacromolecules.
Functionalization Figure 3 shows two ways to anchor molecules on the surface of nanoparticles. In route A, a monolayer7 of molecules bearing a reactive group grows on the nanoparticles; specific molecules then react with the monolayer’s reactive groups to yield the functional nanoparticles. In route B, the group that reacts with the surface is joined first to the functional molecule, then the combination reacts with the nanoparticle to yield the desired product. Route A is simple and versatile, but it may leave unreacted sites as defects. Route B produces a well-defined monolayer that has fewer defect sites; however, it can require considerable synthetic effort to engineer the molecules to react with the surface.
Figure 3. Two ways to attach molecules to a nanoparticle (the red fragment represents the surface anchor; the green one, the reactive group to be attached to the nanoparticle).
Applications for multifunctional magnetic nanoparticles
Capturing bacteria. The ability to detect bacteria at ultralow concentrations without time-consuming procedures is very valuable in clinical diagnosis and environmental monitoring. Doing so using conventional techniques requires an “induction time” of at least 24 hours during the analysis. The use of multifunctional magnetic nanoparticles, as illustrated in Figure 4, can speed this process. After a ligand such as vancomycin [Van] is attached to the surface of the magnetic nanoparticles (e.g., iron-platinum), the magnetic nanoparticles (written as FePt@Van conjugates) become attached to the bacteria, because vancomycin binds to receptors the bacteria carry on their outer membranes—even when the bacteria are present in very low concentrations. A small magnet attracts and enriches the bacteria-nanoparticle composites for analysis. This process takes less than two hours and detects bacteria with very high sensitivity (probably because the FePt@Van nanoparticles are roughly the same size as antibodies, which have evolved to destroy bacteria).
In addition, using functional magnetic nanoparticles to capture bacteria is particularly useful when the polymerase chain reaction8 technique is not applicable, which may be the case for biological mixtures. Combining FePt@Van magnetic nanoparticles with fluorescent dyes can achieve quick, sensitive, and low-cost detection of bacteria in a patient’s blood, for example.9 Newer strategies based on the same principle detect bacteria by anchoring bacteria-specific antibodies to the magnetic nanoparticles.10
Figure 4. (A) Interaction between iron-platinum nanoparticles conjugated with vancomycin (FePt@Van) and bacteria (Staphylococcus aureus). The magnet (bottom illustration) attracts the particles and hence the bound bacteria. (B ) There is no interaction with nanoparticles bearing amine groups (NH2) as the control and so the bacteria are not removed by a magnet. Scanning electron microscope (SEM) images of (C) aggregates of *S. aureus* and FePt@Van nanoparticles, and (D) aggregates of FePt-NH2 nanoparticles. (E) SEM and (F) transmission electron microscope (TEM) images of a VanA bacteria captured by FePt@Van nanoparticles (inset, high resolution TEM image of FePt@Van nanoparticles). Vancomycin-resistant enterococcus (VRE) is the name given to a group of bacterial species of the genus Enterococcus that is resistant to the antibiotic vancomycin. VanA is one of the six different types of vancomycin resistance shown by enterococcus. VRE(VanA) is resistant to vancomycin and teicoplanin. (Reproduced with the permission from ref. 11. ©2003, American Chemical Society).11
Purifying proteins. Studying and developing uses for proteins requires different forms of proteins to be purified and manipulated, which can be challenging because the differences between the forms may be small. Using magnetic nanoparticles to separate the forms can allow exceptionally high selectivity.12,13 For example, we have used magnetic nanoparticles that are linked to nitrilotriacetic acid, a compound that holds onto certain metal ions, to separate protein variants produced in genetically altered bacteria. The proteins were made bearing special chemical tags that bind strongly to the metal ions.14,15 Using nanoparticles achieves much higher specificity in separating variant forms of the protein than using microparticles (which are roughly a thousand times bigger). The high surface-to-volume ratios of nanoparticles, together with their ability to disperse easily, also increases their value for separating mixtures and can simplify analytical procedures. In addition, such magnetic nanoparticles are reusable.
Localizing fluorescent nanomagnets inside a cell. For biological applications, multifunctional nanomaterials have the unique advantage that they can combine superparamagnetism and fluorescence. This means researchers can use magnetic force to control the movement of such particles, which are termed hybrid nanoparticles, within cells, and use microscopes to observe them.16 Figure 5 shows the movement of such nanoparticles upon applying a magnetic force. Without the magnetic force, the nanoparticles evenly distribute inside the cells due to the lack of specific interactions (Figure 5E). After being attracted by a small magnet, the nanoparticles aggregate on the side of the cell nearest the magnet (Figure 5F). This demonstration of intracellular manipulation of magnetic nanoparticles suggests they could be a useful tool in investigating the different sides of polarized cells. To realize this potential, however, the fluorescent magnetic nanoparticles should have a fast response to the magnetic force, and improvement is still needed.
Figure 5. Intracellular manipulation of iron oxide-cadmium selenide nanopoarticles. (A) Distribution of the nanoparticles in a cell without a magnetic field. (B) Distribution of the nanoparticles with the magnetic field (H represents magnetic field and F represents magnetic force). (C) Aggregation due to strong magnetic dipolar-dipolar interactions (FD-D). (D) The aggregates of the nanoparticles inside the cells. The confocal images are of HEK293T cells after being incubated with the nanoparticles and a vector (E) without and (F) with a magnetic field (Reproduced with permission from ref. 16. ©2008, American Chemical Society).16
Summary and challenges
Multifunctional magnetic nanoparticles with high selectivity and high sensitivity not only show promise in the biological applications of bacterial detection and protein purification, they also offer advantages in tumor targeting and multimodal imaging (use of multiple types of imaging simultaneously). Hybrid magnetic nanoparticles open up new avenues for a variety of biomedical applications because of their integrated functions. These two approaches could lead to a wide variety of applications (Figure 6) that are currently under intensive exploration. They include localization of molecules of interest through specific binding to nanoparticles, drug delivery, use with magnetic resonance imaging for research and diagnosis, bacteria detection, protein separation, and use with multimodal imaging. In many of these applications, the ability of nanoparticles to form multiple bonds to a variety of substances of our choosing, and thus to bind tightly where we want them to, is essential to what makes them so uniquely promising.
Figure 6. The scheme illustrates two strategies for fabricating multifunctional magnetic nanoparticles, and their potential applications (QDs represents quantum dots) (Reproduced with permission from the American Chemical Society ©2009).
Despite these exciting potential biomedical applications, challenges and issues remain. For example, heterogeneity of nanomaterials is a major problem and it is still hard to precisely control the number of functional molecules on the surface of nanoparticles. Researchers must develop better strategies for producing nanoparticles that have precise composition, uniform surface modification, and reproducible functionalization. For in vivo biomedical applications, the purity, dispersity, and stability of the multifunctional magnetic nanoparticles in a physiological environment are highly important.17 It is essential, therefore, to further study and explore the properties of these promising bionanomaterials. We have good reasons to believe that research can create a successful nanobiotechnology that will usher in important biomedical advances.
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