Applications of Nanoparticles in Biology

Applications of Nanoparticles in Biology

(Parte 1 de 5)

DOI: 10.1002/adma.200703183

Applications of Nanoparticles in Biology**

By Mrinmoy De, Partha S. Ghosh, and Vincent M. Rotello*

1. Introduction

The use of nanomaterials in biotechnology merges the fields of material science and biology. Nanoparticles provide a particularly useful platform, demonstrating unique properties with potentially wide-ranging therapeutic applications.[1] The field of nanoparticles in biology is certainly a burgeoning one, withtheestimatednumberofpapersinthearea(basedon Web of Science) rising from 1 in 1991 to nearly 10000 in 2007. Clearly, we cannot exhaustively cover the field, so this Review provides a brief overview of recent studies using spherical nanoparticles with metallic, metal oxide, semiconductor, and silica cores.

The unique properties and utility of nanoparticles arise from a variety of attributes, including the similar size of nanoparticles and biomolecules such as proteins and polynucleic acids. Additionally, nanoparticles can be fashioned with a wide range of metal and semiconductor core materials that impart useful properties such as fluorescence and magnetic behavior.[2] The applicable properties of some well-known core materials and corresponding possible ligands used for surface functionalization with their possible applications are summarized in Table 1.

In this Review we will discuss general approaches to the integration of nanoparticles with biomolecules. We will then discuss three major areas of nanoparticle application: (i) drug and gene delivery, (i) biosensing, and (ii) bioimaging.

2. Nanoparticle–Biomolecule Interactions

Biomacromolecule surface recognition by nanoparticles as artificial receptors provides a potential tool for controlling cellular andextracellular processesfor numerous biologicalapplications such as transcription regulation, enzymatic inhibition, delivery and sensing. The size of nanoparticle cores can be tuned from 1.5nm to more than 10nm depending on the core material, providing a suitable platform for the interaction of nanoparticles with proteins and other biomolecules (Fig. 1).[3]

The conjugation of nanoparticles with biomolecules such as proteins and DNA can be done by using two different approaches, direct covalent linkage and non-covalent interactions between the particle and biomolecules.[4–9] The most direct approach to the creation of integrated biomolecule– nanoparticle conjugates is through covalent attachment.[10] This conjugation canbeachievedeither throughchemisorption of the biomolecule to the particle surface or through the use of heterobifunctional linkers. Chemisorption of proteins onto the surface of nanoparticles (usually containing a core of Au, ZnS, CdS, and CdSe/ZnS) can be done through cysteine residues that are present in the protein surface (e.g., oligopeptide, serum albumin),[1] or chemically using 2-iminothiolane (Traut’s reagent).[12] Bifunctional linkers provide a versatile

The wide variety of core materials available, coupled with tunable surface properties, make nanoparticles an excellent platform for a broad range of biological and biomedical applications. This Review provides an introduction to nanoparticle–biomolecular interactions as well as recent applications of nanoparticles in biological sensing, delivery, and imaging of live cells and tissues.

[*] Prof. Vincent M. Rotello, M. De, P. S. Ghosh

Department of Chemistry University of Massachusetts 710 North Pleasant Street Amherst, MA 01003 (USA) E-mail:

[**] The NIH (GM077173) and NSF (Center for Hierarchical Manufacturing, DMI-0531171) are acknowledged.

means of bioconjugation. Biomolecules are often covalently linked to ligands on the nanoparticle surface via traditional coupling strategies such as carbodiimide-mediated amidation and esterification.[13] For biological applications oligoethylene glycol (OEG) or polyethylene glycol (PEG) is used in the linker to enhance thestability of the attached biomolecules and minimize non-specific adsorption of other materials.

Non-covalent assembly provides a highly modular approach to the biofunctionalization of nanoparticles. DNA–NP binding can be effected through electrostatic interactions, groove binding, intercalation, and complementary single-strand DNA binding.[14] Nanoparticles provide an attractive receptor for nucleic acids, providing a direct analogy to protein–DNA interactions.[15,16] One approach to particle–DNA assembly uses complementary electrostatic interactions to promote high affinity of nanoparticle–DNA binding. The use of cationic ligands on the nanoparticle surface provides a complementary surface for binding the negatively charged backbone of DNA, for example the use of NP1 to recognize a 37-mer DNA duplex (Fig. 2a and b) by Rotello.[17] The binding of the DNA inhibited transcription by T7 RNA polymerase, indicating the high affinity of the NP–DNA complex, and pointing out a

M. De et al./Applications of Nanoparticles in Biology

Mrinmoy De received his B.Sc. in Chemistry from Vidyasagar University, India in 2000 and M.Sc. in Organic Chemistry from the Indian Institute of Technology-Bombay, India in 2002. He is currently pursuing a Ph.D. at the Department of Chemistry, University of Massachusetts at Amherst, USA under the guidance of Prof. Vincent M. Rotello. His current research is focused on the use of nanoparticles for biomolecular recognition, their applications in biology, and the development of biomaterials.

Partha S. Ghosh received his B.Sc. in Chemistry from Ramamkrishna Mission Vidyamandira at Belur, India in 2002. He received his M.Sc. from the Indian Institute of Technology-Kanpur, India in 2004. He is currently a graduate student at the Department of Chemistry, University of Massachusetts at Amherst, USA under the guidance of Professor Vincent M. Rotello. His current research is focused on the use of nanoparticles for delivery applications.

Vincent M. Rotello received his B.Sc. from the Illinois Institute of Technology, USA in 1985, his Ph.D. in 1990 from Yale University, USA and was an NSF postdoctoral fellow at M.I.T., USA from 1990–1993. Since 1993, he has been at the University of Massachusetts at Amherst,USA where he is currently the Charles A. Goessmann Professor of Chemistry. His research focuses on the application of synthetic chemistry to polymers, nanotechnology, and biological systems.

Table 1. Characteristics, ligands and representative applications for various metal and semiconductor materials.

Core material Characteristics Ligand(s) Applications

Au Optical absorption, fluorescence and fluorescence quenching, stability Thiol, disulfide, phosphine, amine Biomolecular recognition, delivery, sensing

Ag Surface-enhanced fluorescence Thiol Sensing Pt Catalytic property Thiol, phosphine, amine, isocyanide Bio-catalyst, sensing CdSe Luminescence, photo-stability Thiol, phosphine, pyridine Imaging, sensing

Fe2O3 Magnetic property Diol, dopamine derivative, amine MR imaging and biomolecule purification SiO2 Biocompatibility Alkoxysilane Biocompatible by surface coating

potential use of these systems in gene regulation. Intercalation providesanother mechanismforDNA binding,as demonstrated by Murray et al.[18] A third approach to DNA conjugation exploits the high affinity and specificity of DNA–DNA interactions (Fig. 2c).[19] This approach is discussed further in the biosensor portion of this Review.

Nanoparticle–protein interactions can regulate multiple biological processes such as protein–protein interactions, protein–nucleic acid interactions, and enzyme activity. As with DNA, electrostatic assembly provides a direct means of conjugation. One system that has been explored is the binding of a-chymotrypsin (ChT), exploiting the ring of cationic residues around active site of ChT (Fig. 3).[20] Time-dependent inhibition of ChT activity was observed upon incubation with negatively charged NP 2.[21] A two-step binding process with a fast reversible association followed by a slower irreversible denaturation was established.[2] This interaction could be reversed using cationic surfactants (Fig. 3b), restoring ChT activity.[23] Based on the dynamic light scattering (DLS) data two distinct mechanisms were postulated: alkyl surfactants 3 and 4 form a bilayer structure, whereas cationic thiol 5 and alcohol 6 directly modify the monolayer to liberate the bound proteins.

The use of simple alkyl-based monolayers generally results in protein denaturation; an unfavorable outcome for a number of applications in delivery and biotechnology. Relying on the resistance of OEG to nonspecific interactions with biomolecules,[24] tetra(ethylene glycol) spacers were introduced at the nanoparticle–protein interface.[25] Structural data obtained from fluorescence and circular dichroism (CD) studies revealed thatthenanoparticle-boundChTremainedwithnativestructure. Further studies demonstrated that nanoparticle–protein complexation can considerably stabilize the bound proteins against denaturation at the air/water interface.[26] Specific biomacromolecular interactions such as streptavi- to provide specific protein–NP binding. Zheng and Huang introduced biotin and glutathione-functionalized gold nanoparticles functionalized with tri(ethylene glycol)-terminated thiols.[28] These particles bind specifically to streptavidin and glutathione-S-transferase, respectively, to give stable complexes with minimal nonspecific binding with other proteins (Fig. 3d). Biotin functionalized quantum dots (QDs) were also used for specific protein binding in a time-resolved fluoroimmunoassay.[29] Another way to specifically bind proteins is through the use of transition metal complexes that can bind with surface-exposed histidines of proteins.[30] Xu et al. fabricated FePt magnetic nanoparticles NP 7, with nickelterminated nitrilotriacetic acid (NTA).[31] These NPs show high affinity and specificity towards histidine-tagged proteins (proteins with six consecutive histidine residues) (Fig. 3e). In comparison to commercial magnetic microbeads, these NPs have a great protein binding capacity owing to their high surface-to-volume ratio. This concept can be employed to manipulate the histidine-tagged recombinant proteins and bind other biological substrates at low concentrations.[32,3]

In analogy to proteins, nanoparticles can be used as a multivalent receptor to enhance low-affinity interactions such as carbohydrate–protein interactions.[34] As an example, Lin et al. prepared mannose-functionalized gold nanoparticles and investigated their interaction with the lectin, concanavalin A (Con A).[35] These nanoparticles showed a high affinity to Con

A with a Ka of 107–108dm3 mol 1; an affinity 10-100-fold higher than that of monovalent mannose ligands, an approach that has been used for the sensing of agglutinin proteins.[36–38]

3. Nanoparticles in Biosensing

The sensing of biological agents, diseases, and toxic materials is an important goal for biomedical diagnosis, forensic analysis, and environmental monitoring.[39] A sensor generally consists of two components: a recognition element for target binding and a transduction element for signaling the binding event. The unique physicochemical properties of NPs[9] coupled with the inherent increase in signal-to-noise ratio provided by miniaturization[40] makes these systems promising candidates for sensing applications.[41] As an example, gold nanoparticles exhibits unique optical and electronic properties based on size and shape. Gold nanoparticles show an intense absorption peak from 500 to 550 nm[42] arising from surface plasmon resonance (SPR).[43–45] SPR occurs from the collective

M. De et al./Applications of Nanoparticles in Biology

Figure 1. Schematic representation of a 2nm gold nanoparticle with 1-mercaptoundecanoic acid monolayer and relative sizes of papain and a 24-mer DNA duplex.

Figure 2. The DNA-nanoparticle interactions. a) Structure of NP1 scaffold and the DNA backbone. b) Transcription level as a function of DNA–NP1 stoichiometry. c) Binding of DNA through complementary oligonucleotide hybridization.

Adv. Mater. 2008, 20, 4225–4241 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 4227

oscillation of the conductive electrons owing to the resonant excitation by the incident photons, although the fundamental physical principles of SPR are very complex. The SPR band is sensitive to the surrounding environment, signaling changes in solvent and binding. A particularly useful output is the red-shift (to ca. 650nm) and broadening of the plasmon band due to the interparticle plasmon coupling.[46]Thisphenomenon leadstothe popular and widely applicable colorimetric sensing. Metallic nanoparticles also possess superb quenching ability[47,48] and photoluminescence[49–51] under certain conditions.

3.1. Colorimetric Sensing

The oligonucleotide-mediated nanoparticle aggregation process has been extensively used for the development of simple and highly sensitive colorimetric biosensors for oligonucleotides by Mirkin[52,53] and others.[54–56] The detection of specific oligonucleotide sequences is now very important in diagnosis of genetic and pathogenic diseases and quantifying the amount of product generated by polymerase chain reaction (PCR). The general procedure for detection of oligonucleotides is through the fabrication of nanoparticles, functionalized with single-stranded DNA. Upon addition of the target sequence the particles aggregate, changing the color of the solution (Fig. 4a). Using this method oligonuceotides were detected at sub-picomolar level without the assistance of PCR.[53] This methodology was also applied for the colorimetric screening of DNA binders[57] and triplex DNA binders (Fig. 4b).[58]

Based on the similar approach a highly selective and sensitive lead (Pb2þ) biosensor was reported by Lu et al.[59,60] In their sensor design, they used a Pb2þ-specific ‘‘DNAzyme’’

M. De et al./Applications of Nanoparticles in Biology

Figure 3. Protein–nanoparticle conjugation and its applications. a) Electrostatic targeting of ChT by anionic NP 3 b) Complexation of ChT with anionic nanoparticles and its release mechanisms by addition of various surfactants. Addition of cationic alkyl surfactant (3 and 4) forms a bilayer structure, whereas addition of cationic thiol and alcohol (5 and 6) amends the monolayer. c) Different degree of restoration of enzymatic activity of nanoparticlebound ChT by addition of various positively charged surfactants. d) Specific interaction of biotin-functionalized nanoparticles with streptavidin. e) Structure of NTA-modified magnetic nanoparticles. f) The NTA–Ni2þ functionalized magnetic nanoparticles selectively bind to histidine-tagged proteins.

composed of a catalytic and a substrate strand. In the presence of Pb2þ, the substrate strand cleaves into two pieces (Fig. 5a), resulting in head-to-tail (Fig. 5b) or tail-to-tail (Fig. 5c) aggregation with a concomitant red to blue color shift with a sensing limit of 100nM, which is unaffected by other divalent metal ions.

Anotherattractivesensorapproachusesaptamers, single-stranded oligonucleic acid-based binding molecules that can bind a wide range of targets with high affinity and specificity.[61] An example is cocaine sensing, using a cocainespecific aptamer (Fig. 6).[62] The sensor features a particle functionalized with two different sequences of single-stranded DNA, one for conjugationandanotherwiththecocaineaptamer. In the presence of cocaine the nanoparticles are deaggregated, with a concomitant blue-to-red color change. This method has been extended to mixed-aptamer systems that respond to combinations of analytes,[63,64] as well as the detection of other biomolecular systems such as platelet-derived growth factors (PDGFs)[65] and thrombin.[6]

Nanoparticles featuring ligands targeted at specific biomolecules provide another avenue for the colorimetric detection of proteins. The bivalent lectin agglutinin specifically recognizes b-D-galactose, inducing the aggregation of galactose-functionalized nanoparticles at 1ppm.[67] Other glyconanoparticles have been used for sensing various proteinssuch as Concanavalin A and cholera toxin.[68,69]

Dithiols such as C- and N-terminal cysteinyl peptides can serve as bridging agents to assemble nanoparticles, and have been used for the colorimetric detection of proteases. In a representative study, Stevens et al. reported a two-stage approach by using Fmoc-protected peptides (substrate of thermolysin) with a cysteine amino acid attached to gold nanoparticles.[70] In presence of thermolysin the peptides are fragmented and the assembly changes color from blue to red with a sensitivity of 90zg mL 1 (i.e., less than 380 molecules of protease).[71] This approach has been extended to kinases[72] and phosphatases[73,74] at low concentration.

3.2. Fluorescence Sensing

The exceptional quenching ability of metallic nanoparticles makes them excellent materials for Forster resonance energy transfer (FRET)-based biosensors,[47] for example, for the fabrication of molecular beacons for sensing DNA.[75] In this approach, the dye molecule is close to the nanoparticle surface in the absence of the target DNA strand due to hairpin structure of the attached DNA, resulting in fluorescence quenching (Fig. 7a).Hybridization of the targetDNA opensup the hairpin structure, resulting in a significant increase in

M. De et al./Applications of Nanoparticles in Biology

Figure 4. Schematic illustration of a) DNA-induced nanoparticle aggregation, and b) sensing of DNA triplex binder using DNA-directed AuNP assembly.

(Parte 1 de 5)