Metallic Nanoparticles in Biomedical Applications | Nanobiotechnology

Let us make an in-depth study of the metallic nanoparticles in biomedical applications. The below given article will help you to learn about the following things:- 1. Introduction to Metallic Nanoparticles in Biomedical Applications 2. Application Potential of Silver Nanoparticles in Biological Sciences 3. Gold Nanoparticles as Impor­tant Biomedical Tool and 4. Other Biomedically Important Nanoparticles.

Introduction to Metallic Nanoparticles in Biomedical Applications:

In recent years research involving NPs has generated a great deal of interest from scien­tists and engineers of nearly all disciplines. Among different nanomaterial’s employed for biomedical research, metallic NPs have proved to be the most convenient and suit­able. Based on their unique optical, electrical, magnetic properties, specific heats, melting points, and surface reactivates, metallic NPs have found significant applications in a wide spectrum of biomedical utilities like imaging, sensing, drug delivery and gene targeting.

As all the properties of metallic NPs are size and shape dependent, methods for their prepara­tion is one of the primary thrust areas of re­searchers. Nanostructured metal colloids have been obtained by ‘top-down’ and ‘bottom-up’ ap­proaches. A typical ‘top-down’ is physical method involving the mechanical grinding of bulk metals and subsequent stabilization of the resulting Nano sized metal particles by the addition of colloidal protecting agents.

The ‘bottom-up’, i.e., chemical methods of wet chemical nanoparticle preparation rely on the chemical reduction of metal salts, electrochemical pathways, or the controlled decomposition of metastable organometallic compounds. A large variety of stabilizers, e.g. donor ligands, polymers, and surfactants, are used to control the growth of the primar­ily formed Nano clusters and to prevent them from agglomerating.

As this article is concerned about bio­medical or biological applications of metal­lic NPs, we do not have scope to describe all the existing methods for preparation of metal NPs. This section will be restricted to synthesis of biocompatible NPs, which can be easily administered under in vivo condi­tions. In this context, some major advances have been made by employing methods based on chemical reactions in solution (of­ten termed ‘wet chemistry’).

A wet chemical procedure involves growing nanoparticles in a liquid medium containing various reactants, in particular reducing agents, e.g., Sodium borohydride or potassium bi-tartrate or methoxypolyethylene glycol or hydrazine. Generally in these methods, glucose or some biocompatible reagents are used as reducing agents in place of strong chemical reluctant. Stabilizing agents, e.g., donor ligands, polymers, and surfactants are often employed to prevent NPs from agglomeration and are such that they are easily miscible under cellular conditions.

A surfactant is a molecule that is dynamically absorbed to the surfaces of the NPs under the reaction conditions. It must be mobile enough to provide access for the addition of monomer units, while stable enough to present the aggregation of NPs. The choice of surfactants varies from case to case: a molecule that binds too strongly to the surface of the NPs is not suitable, as it would not allow the NPs to grow. On the other hand, a weakly coordinating molecule would yield large particles, or aggregates.

Some examples of a suitable surfactant or stabilizing agent in­cludes, for instance, alkyl, phosphine’s, phosphine oxides, phosphates, amides or amines, carboxylic acids, sodium dodecyl benzyl sul­fate or polyvinyl pyrrolidone.

As most of the surfactants used are not compatible to cells and tissues, bovine serum albumin is a popular choice to use as stabilizing agent. Recent­ly, scientists have endeavoured to make use of microorganisms as possible eco-friendly Nano-factories for the synthesis of metallic NPs such as cadmium sulfide, gold, and silver. We shall discuss in detail about this biogenic synthesis of various metallic Nano materials in their respective sections.

In recent years, metallic NPs and their al­loys have been studied extensively in various fields like sensor technology, optical devices, catalysis, biological labelling, and drug delivery system, and treatment of some cancers. Me­tallic NPs are quite suitable as a marker for the optical detection of biomolecules in ap­plications such as antimicrobial, antiplatelet, stabilization of proteins, drug delivery or in photo thermal therapeutic applications, due to their excellent SPR properties. These are the extremely promising prospects in the field of health and medicine.

This article provides the researchers a comprehensive present status of the field and points them to other appropriate opportuni­ties where this metallic nanomaterial’s can be employed to cater to unmet biomedical goals. Here we discuss various types of NPs (Table 1) which are belonging to different groups of periodic table and their applica­tions in biomedical fields.

This table gives the overview of some metals that exist in form of Nano powder and their colloidal state:

Application Potential of Silver Nanoparticles in Biological Sciences:

Nano silver particles are generally smaller than 100 nm and contain 20-15,000 silver atoms. Silver NPs have been receiving considerable attention as a result of their unique physical, chemical, biological properties and their im­portant applications in optics, electronics and biomedicine.

Hence, there is a growing need to develop environmentally benign NPs syn­thesis process that does not use toxic chemi­cals and involves environment-friendly pro­cedures for the synthesis and assembly of NPs. Silver NPs are synthesized through chemical method because of their simplicity and cost effectiveness that involves chemical reduc­tion of silver ions in aqueous solutions with or without stabilizing agents, thermal decom­position in organic solvents.

In this context, some major advances have been made by em­ploying methods based on chemical reactions in which citric acid, glucose or some biocompatible reagents are used as reducing agents in place of strong chemical reductants.

Sta­bilizing agents, e.g., donor ligands, polymers, and surfactants are often employed to prevent NPs from agglomeration. In earlier reports we have successfully synthesized biocompatible NPs with enhanced stabilization having anti­bacterial property by using glucose as reduc­ing agent and bovine serum albumin as sta­bilizing agent.

As mentioned above biogenic process involving microorganisms is another approach for biosynthesis of biocompatible silver NPs. Several attempts have been made in this direction. Group of scientists have shown that bacterium Pseudomonas stutzeri AG259, isolated from silver mine, and played ma­jor role in the reduction of the Ag⁺ ions and the formation of silver NPs (AgNPs) when placed in a concentrated aqueous solution of silver nitrate.

AgNPs of well-defined size and distinct topography are formed within the periplasmic space of the bacteria. Another group of scientists have synthesized the silver NPs by using fungus Fusarium oxysporum and Staphylococcus aureus, respectively. Re­cently researchers have successfully synthe­sized AgNPs having synergistic effect against gram +ve and gram -ve bacteria.

NPs of silver have aptly been investigated for their antibacterial property. Commend­able efforts have been made to explore this property using electron microscopy, which has revealed size-dependent interaction of silver NPs with bacteria.

NPs of silver have thus been studied as a medium for antibiotic delivery, and to synthesize composites for use as disinfecting filters and coating materials. However, the bactericidal property of these NPs depend on their stability in the growth medium, since this imparts greater retention time for bacterium-nanoparticle interaction. There lies a strong challenge in preparing NPs of silver stable enough to significantly restrict bacterial growth.

In earlier report we showed the synthesis of highly stable NPs of silver endowed with significant antibacterial prop­erties. Efforts have been made to understand the underlying molecular mechanism of such antimicrobial actions.

In this report we have for the first time shown that silver NPs can bring about changes in tyrosine phosphoproteome of bacteria, thus impacting bacterial cell signalling. Besides their innate antibac­terial property, Nano silver could have many other applications in areas such as non-linear optics, spectrally selective coating for solar energy absorption an intercalation materials for electrical batteries, effectively used for improving the conductivity of electronically conductive adhesive (ECAs), as optical recep­tors and bio-labelling.

In biomedical applica­tions, it was reported that silver NPs in a size range 1-10 nM bind to HIV-1 in a size-de­pendent fashion. The researchers have shown that silver NPs inhibit HIV-1 infection in CD4+MT-2 cells and cMAGI HIV-1 reporter cells.

At present, use of silver is re-emerging as a viable treatment for infections encoun­tered in burns. More recently, silver has also been used as a biocide to prevent infection in burns, traumatic wounds and diabetic ulcers. Other uses include improved surface coating for indwelling catheters and other medical devices implanted on/within the body.

Recently we have for the first time showed that, Nano silver has innate anti-platelet prop­erty and can effectively prevent integrin mediated platelet responses, both in vivo and in vitro, in a concentration-dependent man­ner.

Our findings further suggest that these NPs do not confer any lytic effect on plate­lets and thus hold potential to be promoted as anti-platelet/antithrombotic agent after careful evaluation of toxic effects. Thus, use of Nano silver is becoming more and more widespread in medicine and related appli­cations and—due to increasing exposure— toxicological and environmental issues need to be raised. There has been a continuous de­bate on the advantages and disadvantages of the use of silver products in health care and medicine.

Probably one of the most reported side effects of silver products is argyria. Argyria occurs when sub-dermal silver deposits in skin micro vessels resulting in an irrevers­ible gray to black coloration of the skin. This permanent discoloration is not physically harmful but remains an inherent serious cos­metic problem. There are also some reports of kidney toxicity and cytotoxicity.

Liver tox­icity has also been observed following acute silver toxicity due to Nano crystalline silver. Therefore, understanding the kinetics and toxicity of silver NPs in vivo is very impor­tant in the context of the underlying medical debate regarding the safety of Nano silver and nanomaterial’s coated with silver.

There are several reports that clearly demonstrate that silver, in minute concentrations, is non-toxic to human cells. The epidemiological history of silver has established non-toxicity in nor­mal use. Thus, it is our opinion that these are questions that need to be imperatively an­swered before people rush to indulge into the Nano silver boom.

Gold Nanoparticles as Impor­tant Biomedical Tool:

In past decades, gold NPs have attracted a continuous interest and have been explored as a model platform for biomedical research because of their unusual but unique physical and chemical properties. Au particles are in­ert, which makes them relatively more biocompatible. The synthesis of gold NPs with diam­eters ranging from a few to several hundreds of nanometers is well-established in aqueous solution as well as in organic solvents.

Like silver NPs, gold salts as HAuCl₄ are reduced by the addition of a reducing agent which leads to the nucleation of Au ions to NPs. Turkevitch et al (1951) for the first time syn­thesized the colloidal gold Au⁰ from Au¹¹¹ by using citric acid as reducing agent, a method that is still used to subsequently replace the citrate ligand of these AuNPs by appropriate ligands of biological interest.

Recent modi­fications of the Turkevitch method have al­lowed better size distribution and size con­trol within the 9-120 nm range. In addition, stabilizing agents are also required which are either adsorbed or chemically bound to the surface of the AuNPs.

These stabilizing agents (often also called surfactants) are typi­cally charged, so that the equally charged NPs repel each other so that they are colloidally stable. Although AuNPs can be stabilized by a large variety of stabilizers (ligands, surfac­tants, polymers, dendrites, biomolecules, etc.) the most robust AuNPs were thox that can be stabilized by thiolates using the strong Au-S bond between the soft acid Au and the soft thiolate base.

Along this line, by far the most popular synthetic method using such sulfur coordination for AuNP stabilization is the Shiffrin-Brust biphasic synthesis using HAuCl₄, a thiol, tetractlyammonium bromide and NaBH₄ in water-toluene yielding thio-late-AuNPs.

Functional thiolates can also be introduced using this method or upon subse­quent bio-molecular substitution of a thiolate ligand by such a functional thiol. Oligonucle­otides, peptides and Polyethylene glycol are easily attached to AuNPs in this way.

Since the solubility of these AuNPs is controlled by the solubilizing properties of the termi­nal group of the thiolate ligands, AuNPs can be transferred from an aqueous phase to an organic phase or vice versa by appropriate ligand exchange. Water-soluble AuNPs typi­cally contain terminal carboxylate groups at their periphery.

The carboxyl group is used to attach the amino groups of biomolecules us­ing l-ethyl-3(3-dimethylaminopropyl)-car- bodimide-HCl (EDC). With related strategies almost all kinds of biological molecules can be attached to the particle surface.

Though such protocols are relatively well-established, bio conjugation of Au nanoparticles still is not trivial and characterization of synthe­sized conjugates is necessary—in particular to rule out aggregation effects or unspecific binding during the conjugation reaction.

In particular, in many conjugation protocols, the number of attached molecules per gold nanoparticle is only a rather rough estimate, as no standard method for determining the surface coverage of particles modified with molecules has yet been established.

Very interestingly, not only spherical AuNPs but also NPs bearing various shapes and geometries such as rod or hollow shells can also be synthesized by using appropriate techniques. Au nanoparticles have been pri­marily used for labelling applications.

In this regard, the particles are directed and enriched at the region of interest and they provide con­trast for the observation and visualization of this region. Gold particles strongly absorb and scatter visible light. Upon light absorp­tion the light energy excites the free electrons in the Au particles to a collective oscillation, the co-called surface Plasmon.

Generally, the optical properties of small metal NPs are dominated by collective oscillation of elec­trons at surfaces (known as Surface Plasmon Resonance or SPR) that are in resonance with the incident electromagnetic radiation.

For gold, it happens that the resonance frequency of this oscillation, governed by its bulk di­electric constant, lies in the visible region of the electromagnetic spectrum. Because NPs have a high surface area to volume ratio, the Plasmon frequency is exquisitely sensitive to the dielectric (refractive index) nature of its interface with the local medium.

Any change to the environment of these particles (surface modification, aggregation, medium refractive index, etc.) leads to colorimetric changes of the dispersions. Due to coupling of the Plasmon’s, assemblies (or aggregations) of AuNPs are often accompanied by distinct color changes from red (disperse) to blue (ag­gregated).

As the gold nanoparticles provide excellent contrast for TEM imaging with high lateral resolution and are more stable (do not suffer photo bleaching), which is a major limitation for fluorescence based methods, they are wide­ly used in immuno-staining and single parti­cle tracking for visualizing structures within single cells.

Gold nanoparticle has also been used for a long time for delivery of molecules into cells. For this purpose the molecules are adsorbed on the surface of the Au particles and the whole conjugate is introduced into the cells. Introduction into cells can either be forced as in the case of gene guns or achieved naturally by particle ingestion. Inside cells the molecules will eventually detach themselves from the Au particles.

This particle uptake-mediated delivery of molecules into cells is used mainly for two applications.

Firstly, in gene therapy, DNA is introduced into cells, which subsequently causes the expression of the corresponding proteins.

Secondly, in drug- targeting anti-cancer drugs are delivered spe­cifically to cancer tissue. Delivery applications using gold nanoparticles have been reviewed recently. Besides imaging, gold nanoparticles also act as a heat source because the free electrons in the gold particles are excited upon absorption of light.

Excitation at the Plasmon resonance frequency causes a collective oscil­lation of the free electrons. Upon interaction between the electrons and the crystal lattice of the gold particles, the electrons relax and the thermal energy is transferred to the lat­tice.

Subsequently the heat from the gold par­ticles is dissipated into the surrounding envi­ronment. Controlled heating of gold particles can be used in several ways for manipulating the surrounding tissues. Due to the heat re­leased by the gold particles to the surround­ing tissue, cancerous tissues can be destroyed locally (Hyperthermia) without exposing the entire organism to elevated temperatures.

Based upon bio-conjugation ability, surface Plasmon, fluorescence quenching, surface en­hanced Raman scattering and electron trans­fer characteristics, gold nanoparticles have highly been explored in the field of sensor.

Although gold nanoparticles are composed of an inert material and have extraordinary properties, bio-compatibility issues have to be well-addressed. Several groups have exam­ined the cellular uptake and cellular toxicity of gold nanoparticles. Cells exposed to gold nanoparticles will incorporate the can-particles (similar to nanoparticles of other mate­rials) and store inside the cells in perinuclear compartments, vesicular structures close to the cell nucleus.

Due to particle internal­ization, cells or tissues in contact with gold nanoparticles will be exposed to the particles for extended periods of time. Inflammatory effects in tissues caused by gold particles have been demonstrated. However, in cell culture experiments, Au nanoparticles are regarded as bio-compatible and acute cytotoxicity has not been observed so far. In particular, no re­lease of toxic ions as in the case of cadmium nanoparticles has been reported.

On the oth­er hand, there are few examples of toxic ef­fects related to the nature of Au, which might depend on the cell line. For example, 33 nm citrate-capped gold Nano spheres were found to be non-toxic to baby hamster kidney and human hepatocellular liver carcinoma cells, but were cytotoxic to a human carcinoma lung cell line.

Gold cytotoxicity also depends on surface chemistry, and on the particle size. The toxicity of the gold nanoparticles is re­lated to their interactions with the cell mem­brane, a feature initially mediated by their strong electrostatic attraction to the negative­ly charged bilayer.

However, to determine and understand the toxic effects of gold nanopar­ticles, strategies and interpretation of the data must be done correctly and assumptions must be taken into consideration to ameliorate the observed toxicity.

Other Biomedically Important Nanoparticles:

Metallic nanoparticles are among the most widely used types of engineered Nano-materials. Apart from the above-discussed biomedi­cal applications of silver and gold nanopar­ticles, metallic nanoparticles are also used in integration with magnetic nanoparticles. They together form heterodimer structures that of­fer two distinct surfaces and properties to al­low different kinds of functional molecules to attach onto the specific parts of the heterodimers, which may bind to multiple receptors or act as agents for multimodality imaging.

Magnetic nanoparticles are well-established Nano-materials that provide many exciting op­portunities in biomedical applications. They not only deliver controllable sizes ranging from a few up to tens of nanometers, but they can also be manipulated by external magnet­ic force along with enhancement of contrast in magnetic resonance imaging (MRI). As a result, these nanoparticles have been found promising in several applications in biology and medicine, including protein purification, drug delivery, imaging, tagging, sensing and separation in recent years.

Biomedical appli­cations of magnetic nanoparticles can be clas­sified according to their application inside or outside the body. In vivo applications could further be separated into therapeutic (hyper­thermia and drug-targeting) and diagnostic (nuclear magnetic resonance (NMR) imag­ing) applications.

As far as in vitro applica­tions are concerned, their main uses lie in diagnostics (separation/selection and bioassays). Iron and their oxides such as magnetite (Fe_3OO₄) or maghemite (c-Fe₂O₃) are by far the most commonly employed for biomedical ap­plications. They are also used in conjugation with compounds such as dextran or starch as a coating material to increase their further biocompatibility.

Other magnetic Nano-materials such as co­balt and nickel nanoparticles are also used in biomedical field but are of little interest be­cause of their toxicity, susceptible to oxidation. Magnetic nanoparticles, like silver or gold particles, have been frequently synthesized by the reduction of metal salts using reducing agents in the presence of surfactant molecule.

Recently, techniques and procedures for pro­ducing Mono-dispersed and size-controllable magnetic nanoparticles (e.g., FePt, Fe₃O₄, and y-Fe₂O₃) have advanced considerably, which leads to very active explorations of the appli­cations of magnetic nanoparticles, in the field of biomedicine.

One of the simplest and most efficient methods of developing integrated metallic and magnetic nanoparticles is the sequential growth of metallic components (e.g., Ag or Au) onto a ’colloid some’ (i.e. the self-assem­bly of nanoparticles at a liquid-liquid inter­face) of magnetic nanoparticles. The metallic nucleation takes place the exposed surface of the magnetic nanoparticles and produces the heterodimers of two distinct Nano spheres.

Recently a group of researchers reported an­other way to fabricate Fe₃O₄-Au heterodim­ers in a homogeneous organic solvent, where the thermal decomposition of Fe(CO)₅ onto the surface of the Au nanoparticles and the following oxidation of intermediate result in uniform Fe,O-Au heterodimers.

The heterodimer structure offers particles with two distinct surfaces. Different kinds of function­al molecules can covalently bind to the spe­cific parts of the heterodimers. For instance, Fe₃O₄ can support a specific biomolecule for targeting using dopamine as a robust anchor. For the Ag or Au component, one can use the well-developed gold-thiol chemistry for the attachment of thiol-terminated biomolecules.

Together with own distinct functional­ities, these multifunctional heterodimers can respond to magnetic forces, show enhanced resonance absorption and scattering, and bind with specific receptors.

Using epidermal growth factor receptor isoform A (EGFRA)-conjugated Fe₃O₄-Au heterodimer nanoparticles, researchers have demonstrated their dual-functional imaging property for cell tracking. This type of het­erodimer nanoparticle may have great poten­tial in multimodal biomedical applications, especially for molecular imaging, but a sub­stantive amount of work need to be done to achieve these promising applications.