User:Inorganicchemutm/Silver nanoparticles (chemotherapy)

A silver nanoparticle is a composition of silver atoms colloided into a nanometer size particle. A nanoparticle of silver can range from 1 nanometer to100 nanometers in diameter. Numerous shapes of nanoparticles can be constructed depending on the application at hand. Commonly used are spherical silver nanoparticles but diamond, octagonal and thin sheets are also popular. Silver nanoparticles present novel applications in the treatment of various diseases. Their extremely large surface area permits the coordination of a vast number of ligands. The properties of silver nanoparticles that are applicable to human treatments have been recently, and still are, under considerable investigation. Aspects of toxicity, cost and other economic considerations are also the subjects of further discussion.

Synthesis
Several approaches in the synthesis of silver nanoparticles have been noted. Outlined below are the methods most commonly employed by researchers working in the medicinal field of nanoparticle technology to prepare silver nanoparticles for biomedical applications.

Coating with silica
In this method, polyvinylpyrrolidone (PVP) is dissolved in water by sonication and mixed with silver colloid particles. Active stirring ensures the PVP has absorbed to the nanoparticle surface. Centrifuging separates the PVP coated nanoparticles which are then transferred to a solution of ethanol to be centrifuged further and placed in a solution of ammonia, ethanol and Si(OEt4) (TES). Stirring for twelve hours results in the silica shell being formed consisting of a surrounding layer of silicon oxide with an ether linkage available to add functionality. Varying the amount of TES allows for different thicknesses of shells formed. This technique is popular due to the ability to add a variety of functionality to the exposed silica surface.

Biogenic Synthesis
The biological synthesis of nanoparticles has provided a means for improved techniques and methods in the medicinal field. Biogenic methods can be used to make nanoparticles of different chemical compositions, sizes, and shapes without the use of toxic ingredients as used currently in synthetic protocols. In addition, precise control over shape and size is vital during nanoparticle synthesis since the NPs therapeutic properties are intimately dependent on such factors. Hence, the primary focus of research in biogenic synthesis is in developing methods that consistently reproduce NPs with precise properties.

Plant Derived
Interestingly, an alternate method of synthesizing silver nanoparticles comes from the extracts of plant material, commonly referred to as “green synthesis”. Extracts from flowers such as germanium and many pepper species have been used as the initial reducing agent of Ag+ in the first step of nanoparticle synthesis. The extract can be mixed with silver nitrite and treated with distilled water, acetone and ethanol to crystallize the colloid suspension. The resultant silver nanoparticles are synthesized without the use of harmful reagents such as sodium borohydride and sodium citrate.

Alternatives
Bacterial and fungal synthesis of nanoparticles is practical because bacteria and fungi are easy to handle and can be modified genetically with ease. This provides a means to develop biomolecules that can synthesize AgNPs of varying shapes and sizes in high yield, which is at the forefront of current challenges in nanoparticle synthesis. Fungal strains such as Verticillium and bacterial strains such as K. pneumoniae can be used in the synthesis of silver nanoparticles. When the fungus/bacteria is added to solution, protein biomass is released into the solution. Electron donating residues such as tryptophan and tyrosine reduce silver ions in solution contributed by silver nitrate. This again causes colloid crystallization and thus the formation of nanoparticles similar to the previous methods.

Therapeutic Applications
Silver nanoparticles have long been known for their antibacterial, antifungal, anti-viral and anti-inflammatory properties. Recently, researchers have extend their use into chemotherapy as a device for delivering various payloads such as small drug molecules or large biomolecules to specific targets. Once the AgNP has had sufficient time to reach its target, release of the payload could be triggered by internal or external stimulus. The targeting and accumulation of nanoparticles to a designated area ensures high drug concentration at specific sites and thus minimizes side effects. This is important because it eliminates the need for higher drug dosages, which are normally administered during current chemotherapeutic treatments to compensate for drug loss.

Chemotherapy
The introduction of nanotechnology into medicine is expected to greatly advance diagnostic cancer imaging and the standards for therapeutic drug design. Nanotechnology may uncover insight about the structure, function and organizational level of the biosystem at the nanoscale. This translates into new treatments and potential cures for diseases such as cancer, which remains one of the world's most devastating diseases. Current cancer treatments include surgical intervention, radiation and chemotherapeutic drugs, which often cause toxicity to the patient, and death of healthy cells. The use of nanoparticles is at the forefront of projects in current research with the emerging trend of the NP acting as an anti-cancer agent itself in addition to being a drug carrier.

Drug Targeting
As mentioned previously, silver nanoparticles can undergo coating techniques that offer a uniform functionalized surface to which substrates can be added. When the nanoparticle is coated, for example, in silica the surface exists as silicic acid. Substrates can thus be added through stable ether and ester linkages that are not degraded immediately by natural metabolic enzymes. Recent chemotherapeutic applications have designed anti cancer drugs with a photo cleavable linker, such as an ortho-nitrobenzyl bridge, attaching it to the substrate on the nanoparticle surface. The low toxicity nanoparticle complex can remain viable under metabolic attack for the time necessary to be distributed throughout the bodies systems. If a cancerous tumor is being targeted for treatment, ultraviolet light can be introduced over the tumor region. The electromagnetic energy of the light causes the photo responsive linker to break between the drug and the nanoparticle substrate. The drug is now cleaved and released in an unaltered active form to act on the cancerous tumor cells. Advantages anticipated for this method is that the drug is transported without highly toxic compounds, the drug is released without harmful radiation or relying on a specific chemical reaction to occur and the drug can be selectively released at a target tissue.

A second approach is to attach a chemotherapeutic drug directly to the functionalized surface of the silver nanoparticle combined with a nucelophilic species to undergo a displacement reaction. For example once the nanoparticle drug complex enters or is in the vicinity of the target tissue or cells, a glutathione monoester can be administered to the site. The nucleophilic ester oxygen will attach to the functionalized surface of the nanoparticle through a new ester linkage while the drug is released to its surroundings. The drug is now active and can exert its biological function on the cells immediate to its surroundings limiting non-desirable interactions with other tissues.

Multiple Drug Resistance
A major cause for the ineffectiveness of current chemotherapy treatments is due to multiple drug resistance. During the progression of tumors, cancer cells divide, passing their inherently unstable genomes onto their daughter cells. Cumulatively, as the tumor expands, new genomic alterations may arise in daughter cells; changing the properties on the outskirts of the tumor. These changes may include drug impermeability, consequently rendering the chemotherapeutic agents ineffective to those particular cells. Multiple drug resistance occurs when transporter proteins on the surface of the cancer cell are over-expressed; enhancing the expelling of that drug and increasing the development of resistance to the treatment. Expelling chemotherapy drugs inevitably diminishes the therapeutic effect and causes the treatment to fail as resistant cells continue to replicate.

Nanoparticles can provide a means to overcome MDR. In general, when using a targeting agent to deliver nanocarriers to cancer cells, it is imperative that the agent binds with high selectivity to molecules that are uniquely expressed on the cell surface. Hence NPs can be designed with proteins that specifically detect drug resistant cells with overexpressed transporter proteins on their surface. A pitfall of the commonly used nano-drug delivery systems is that free drugs that are released from the nanocarriers into the cytosol get exposed to the MDR transporters once again, and are exported. To solve this, 8nm nano crystalline silver particles were modified by the addition of a trans-activating transcriptional activator (TAT) from the HIV-1 virus which, acts as a cell penetrating peptide (CPP). Generally, AgNP effectiveness is limited due to the lack of efficient cellular uptake; however, CPP-modification has become one of the most efficient methods for improving intracellular delivery of nanoparticles. Once ingested, the export of the AgNP is prevented based on a size exclusion. The concept is simple: the nanoparticles are too large to be effluxed by the MDR transporters, because the efflux function is strictly subjected to the size of its substrates, which is generally limited to a range of 300-2000 Da. Thereby the nanoparticulates remain insusceptible to the efflux, providing a means to accumulate in high concentrations.

Malignant melanoma is one of the most aggressive skin cancers with active metastatic potential and notoriously high resistance to cytotoxic agents, and no effective chemotherapy is clinically available yet. In the in vivo experiments, drug-resistant B16 melanoma cells were transplanted into nude mice. AgNP-TAT or AgNP were peritumorally given to the mice bearing melanoma. The AgNP-TAT NPs were able to effectively inhibit tumor growth in mice bearing malignant melanoma at a dose of 1 nmol/kg, and showed significantly reduced adverse toxicity in vivo. TAT-modifications were achieved through a stable Ag-S bond, in which the TAT peptide, bearing a free thiol group at its N-terminal was anchored to the surface of the AgNP.

Antimicrobial
Introduction of silver into bacterial cells induces a high degree of structural and morphological changes, which can lead to cell death. As the silver nano particles come in contact with the bacteria, they adhere to the cell wall and cell membrane. Once bound, some of the silver passes through to the inside, and interacts with phosphate-containing compounds like DNA and RNA, while another portion adheres to the sulphur-containing proteins on the membrane. The silver-sulphur interactions at the membrane cause the cell wall to undergo structural changes, like the formation of pits and pores. Through these pores, cellular components are released into the extracellular fluid, simply due to the osmotic difference. Within the cell, the integration of silver creates a low molecular weight region where the DNA then condenses. Having DNA in a condensed state inhibits the cell’s replication proteins contact with the DNA. Thus the introduction of silver nanoparticles inhibits replication and is sufficient to cause the death of the cell. Further increasing their effect, when silver comes in contact with fluids, it tends to ionize which increases the nanoparticles bactericidal activity. This has been correlated to the suppression of enzymes and inhibited expression of proteins that relate to the cell’s ability to produce ATP. Although it varies for every type of cell proposed, as their cell membrane composition varies greatly, It has been seen that in general, silver nano particles with an average size of 10nm or less show electronic effects that greatly increase their bactericidal activity. This could also be partly due to the fact that as their size decreases, their reactivity increases (due to the surface area to volume ratio increase) as well, a smaller nano particle would require less effort to enter the cell, where the silver can ionize and further increase its bactericidal activity.

It has been noted that the introduction of silver nano particles has shown to have synergistic activity with common antibiotics already used today, such as; penicillin G, ampicillin, erythromycin, clindamycin, and vancomycin against E. coli and S. aureus. In medical equipment, it has been shown that silver nano particles drastically lower the bacterial count on devices used. However, the problem arises when the procedure is over and a new one must be done. In the process of washing the instruments a large portion of the silver anno particles become less effective due to the loss of silver ions. They are more commonly used in skin grafts for burn victims as the silver nano particles embedded with the graft provide better antimicrobial activity and result in significantly less scarring of the victim. They also show promising application as water treatment method form clean potable water.

Safety and Toxicity
Although silver nanoparticles have been applied frequently in chemotherapeutic treatments, their toxicity to non-diseased cells is often a concern. Several studies have focused on the extent of silver nanoparticle toxicity including different sizes, coating techniques and dosages. Outlines below are examples of the major ways in which silver nanoparticles have posed as toxic agents to mammalian cells. The storage of silver nanoparticles has been shown to increase their toxicity. During storage period of several days at room temperature coated silver nanoparticles can undergo dissolution releasing free silver ions. Kittler et al report using different functionalized silver nanoparticles of sizes ranging from 30 nm to 70 nm. The solvated silver nanoparticles where exposed to cultures of human mesenchymal stem cells to indicate cell viability and toxicity. Reports suggest that the stem cells decreased to 70% viability after being exposed to silver nanoparticles aged for three days and complete cell death observed after a one-month storage period. Freshly prepared silver nanoparticles were used as a control for each trial. The toxicity of silver ions arises from unfavorable binding interactions with DNA/ nucleic acids, mitochondrion, cell wall components and nucleophilic enzyme active site residues. The binding of silver ions to electron rich DNA species can cause double strand breaks. This renders the DNA unable to repair the strands leading to chromosome abnormalities. Dysfunctional cell cycles for replication and division are also exhibited as a result of DNA damage.

The electron transport chain and ATP synthesis takes place within the mitochondrion of the cell. Silver ions can act to block the transport chain or transfer electrons to gaseous oxygen molecules to form toxic superoxides and free radicals. If the electron transport chain is inhibited, the production of ATP is also inhibited. ATP is involved in the mechanisms that repair DNA damage as well as cell cycle checkpoints. Whether DNA is damaged from silver ions or through random mutations, inhibiting ATP synthesis renders the cell unable to repair or replicate its DNA.

Protein channels and nuclear membrane pores can often be in the size range of 9nm to 10 nm in diameter. Small silver nanoparticles constructed of this size have the ability to not only pass through the membrane to interact with internal structures but also to be become lodged within the membrane. Silver nanoparticle depositions in the membrane can impact regulation of solutes, exchange of proteins and cell recognition. In the cell cytosol, an array of enzymes can be the potential target for free silver ions. Silver ions act as lewis acids, accepting electrons from nucleophilic active sites of enzymes. Coordination from a silver ion or multiple silver ions in the enzyme active can hinder the ability for the enzyme to accept its natural substrate and catalyze important biochemical reactions.

Regulation
Contrary to common speculation, not just any chemical research laboratory can pursue work with nanoparticles. Regulations have been put in place requiring all laboratories that work with nanoparticles to be certified to do so. Safety concerns regarding nanoparticles have arisen due to the uncertainties of their health effects. Nanoparticle technology is a novel field of research with many exciting applications. Since it is so recent, minimal testing into the potential health effects on humans and other animals has been done. Some concerns that have been brought up in the human system are the fact that nanoparticles serve as a source of free ions in solution of whatever they are synthesized from. In this case silver nanoparticles are a source of silver ions and being a heavy metal there are concerns of health effects related to the build up of these ions in tissues. Also, if the silver nanoparticle is being used as a carrier for a drug molecule, additional tests need to be carried out to model the interactions of the nanoparticles with other targets in the body besides the one of interest.

Due to the safety concerns, a research laboratory must be certified by the Environmental Health and Safety department (EHS) before commencing in work with nanoparticles. This certification requires that all work with nanoparticles be completed in a fume hood or local exhaust ventilation system that is inspected annually by the EHS. All testing procedures must be examined and approved by the Biological Safety Officer prior to its performance. All other lab safety equipment including safety showers, eyewash and fire extinguishers must be inspected and in working order. A nanomaterial safety course accredited by EHS must also be completed by personal governing work with nanoparticles.