Justyna SIEMIENIEC ? Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wroclaw, Poland
Each traditional drug introduced into the body is causing various side effects. This usually results from low specificity, difficulties in reaching its destination, and fast metabolism. To prevent this, in order to improve the biodistribution of the drug, reduce side effects and thus improve the quality of life of patients, intensive research on the use of nanotechnology developments in medicine is carried out. Drug nanocarriers, metal nanoparticles or polymer capsules are the most commonly used for this purpose. Additionally, to ensure effective treatment, rapid and accurate medical diagnosis is equally important. In this field the quantum dots with their specific optical properties closely connected with the size of the order of nanometers attract specific attention.
Please cite as: CHEMIK 2013, 67, 2, 83-90
In 1959, Professor Richard Feynman at the congress of the American Physical Society delivered a lecture entitled ?There is a plenty of room at the bottom? . He considered the possibility of change of the properties of the material through the manipulation at the level of individual atoms instead at the level of standard chemical synthesis. His lecture and studies initiated the era of nanotechnology, that is a mean of operations on structures smaller than 1?m, by building this size units atom by atom (a trend known as ?bottom-up?) or a miniaturization of the larger systems (so-called ?top-down?) . Currently countless studies on such miniaturized systems are carried out, ranging from electronics and information technology to innovative methods of diagnosis and disease treatment. According to the Science Citation Index report , in years 1984 to 2004 the highest interest has been observed in nanomedicine (76%), especially drug delivery systems (Fig. 1). Other fields of interest are biomaterials, diagnostics and imaging illustrated by 11%, 6% and 4% published papers are respectively (Fig. 1).
Many of the pharmacological properties of conventional drugs can be improved by using innovative systems transporting drugs. They are able to increase the half-life of the active substance, its specificity to the target cells or tissues, to delay its metabolism and to allow the accurate dosage. This will result in a reduction of side effects, particularly severe in the case of long-term therapy, thereby improving quality of life of patients . Nanocarriers can be polymer capsules, liposomes, dendrimers, carbon nanotubes, and metal nanoparticles with suitably functionalized surface . Application of nanocarriers changes the biodistribution of the active substance, causing its higher accumulation in patients? cells. The most useful here are passive or active transport systems. Passive transport (enhanced permeability and retention effect) uses the natural openings in the endothelium. Since the openings between cancer cells are larger (between 400 ? 600 nm)  than these between normal cells, this allows to apply higher concentration of the drug in patients tissues (Fig. 2a). Active transport involves the creation of nanoplatforms, which apart from anchored drug also contain specific antibodies or proteins acting at the target sites (Fig. 2b) . In special cases we can apply a single, non-typical type of transport ? magnetic transport. It is possible when nanocarriers have magnetic properties, with iron oxide nanoparticles being most commonly used. In this case, movement of the nanoparticle ? drug conjugate is dependent on the externally applied magnetic field gradient. When the external magnetic field strength is higher than linear blood flow the nanoparticles are stopped at the target site (Fig. 2b) .
In order to be used in the body nanocarriers should meet specific requirements by being biocompatible, nontoxic and do not accumulating in the tissues. Besides, they should have appropriate functional groups for attachment of the active substance, while maintaining their medicinal properties. To achieve this nanovehicles are often covered with suitably functionalized connectors, which can include proteins, sugars, or polymers . At present, there are already several medicinal substances consisting of carrier and conjugated drug (Tab.1) available at the market, and even more are in various stages of clinical trials .
Among the problems connected with the formation of such conjugates often happens that the medicine delivered in the form bounded to the carrier is not active prior to its release. Therefore, it is essential to release the drug at the destination site. Today, anti-cancer drugs connected to vehicle the most commonly use difference in pH between normal and cancer cells. In healthy cells, the pH is about 7, while the tumor tissue environment it is usually about pH ~ 6 . Another way is to stimulate decomposition of the carrier with UV light at a specific wavelength, such as system used for capsules with silicon oxide. The active substance is placed in the pores closed with the gold nanoparticles to which is attached positively charged linker (ammonium bromide is shown in Fig. 3 ? TUNA). Through electrostatic interactions with negatively charged silica surface, it closes the holes. Upon exposition of a conjugate to UV light, TUNA decays, arises a negatively charged thioundecyltetraethyleneglycolcarboxylate (TUEC) thereby causing release of gold nanoparticles from the silica surface, which results in release of the drug (Fig. 3) .
To close the active substance within the interior of the capsule particles of azobenzene derivatives can also be used because of their ability of cis-trans isomerization under the influence of light with a wavelength of 450 nm . Another method to bind the active ingredient is the use of affinity of gold to the sulfur atom. When presumable drug contains terminal mercapto moiety at both sides of a modified linker, it is possible to connect the drug by creating a disulphide bond then it reductions using dithiothreitol which gives the appropriate form of the transport drug . Next method of the cleavage of the drug ? the carrier bond is to act on this complex with an enzyme specifically present in pathologically changed tissue. The best results have been achieved when the enzyme was specific for a certain disease . Application of appropriate three components of the system, namely drug, carrier and method of drug attachment and release is strongly dependent on the site of drug destination and nature of the treatment of certain disease. These could be defined by appropriate diagnosis.
The effectiveness of the therapy is highly dependent on the diagnosis, its speed and accuracy. Therefore, while the designing new drugs it is simultaneously desirable to create more accurate and faster diagnostic methods. Also in this field nanotechnology has been applied, with the greatest interest being put on magnetic nanoparticles, quantum dots and gold nanoparticles. Every diagnostic method needs appropriate analytical techniques. In the case of nanoparticles the application of surface plasmon resonance  is of key importance, an optical method based on measurement of adsorption on the surface of nanometer metal layer ? usually gold (Fig. 4).
During the flow of analyzed sample through a modified layer of gold attachment of compounds from the analyte to nanogold layer changes optical properties of the layer . Mostly, proteins are analyzed by this method. For example gold surface modified with carboxydextrane allows attachment of specific antigens, which in turn enables measurement of antigen ? antigen interaction. In addition, modification protects the surface from other compounds that may be absorbed, and allows easy regeneration of surface . The development of diagnostic methods, including magnetic resonance imaging facilitate the synthesis of new complexes of gadolinium ions with super-paramagnetic nanoparticles . Interactions between complexes and the surrounding protons decrease the relaxation time in tissues where they are located and thus increase the contrast between the studied areas. In the case of their accumulation in the cells it allows to distinguish pathological from normal tissues . In order to work efficiently, such systems should have the following properties: have a size of about 10 nm to pass through the slits in the membrane, be biocompatible, superparamagnetic (i.e. have zero magnetic moment outside the magnetic field) and have the largest magnetic moment in external magnetic field. Magnetic nanoparticles are also used in diagnostic tests, where the interaction with the analyte generates a magnetic signal that can be measured by magnetometer .
Similar to nanocarriers of drugs, ?diagnostic? nanoparticles are often surrounded by an additional organic or inorganic layer to compensate for possible toxicity. Such a coating may be formed by silica, dextran, chitosan or poly(ethylene glycol).
Also the polymer core complexes can be used as a platform for contrast agents. A perfect example here is a dendrimer with the ethylenediamine core . Due to the presence of high number of amino moieties in its structure it is possible to connect to the dendrimer a variety of chelating agents such as metal ions e.g. Gadolinium. To increase the specificity of the synthesized carrier connection of appropriate vectors ? such as monoclonal antibodies or avidin  has been performed. Increasingly popular both in the medical diagnostics and in drug delivery became inorganic nanocrystals, semiconductor with size of several nanometers, which are related to charge carriers ? both holes and electrons. In 1974, IBM developed the quantum wells, which due to their size have only two dimensions . A few years later, almost at the same time the Laboratory of Texas Instruments  and Bell Labs  published the results of studies on the electron movement freezing in quazizerodimensional quantum dot. When the motion of electrons is reduced in each dimension, there is an energy quantization as it happens in atoms. Hence the other name of quantum dots is: artificial atoms .
Quantum dots (QD)
In contrast to natural atoms, artificially prepared structures described above can have different shapes (from dots, by wire to the disks or cubes) and size (from several to dozens of nanometers). Their size determines their properties, in particular the maximum fluorescence wavelength (Fig. 5) .
Depending on the size and chemical composition, the extent of fluorescence can be from 300 to 2000 nm, covering thereby nearinfrared, visible light and ultraviolet waveband (Fig. 6) .
Their additional advantage is even 100-fold increase in photoluminescence intensity in comparison with organic dyes (fluoresceine, rhodamine) . However, their synthesis is much more complex and more expensive. The most popular are CdSe, CdZn, CdS, ZnSe and ZnS quantum dots. It is possible to create structures of the type core ? shell. Such quantum dots are synthesized by dropwise addition of a solution of Zn(CH3)2/S to the CdSe/TOPO (trioctylphosphine oxide) to give CdSe QDs covered with ZnS thin layer. This extra layer protects the core from oxidation, harsh biological conditions  and enhances the photoluminescence signal. It is worth to note that coated quantum dots are neither biocompatible nor soluble in water. QD-type CdSe/ZnS are usually coated with 3-mercaptopropionic acid because zinc, like gold, has high affinity to the sulfur atoms, moreover, the carboxyl group located at the other end of linker allows subsequent attachment of proteins, aptamers or the appropriate antibodies. However, this method has disadvantages: it reduces the intensity of luminescence and often leads to aggregation of the dots. Another method is to cover QDs with silica layer that directly covers the ZnS layer. The resulting quantum dots are soluble in polar solvents such as methanol or dimethyl sulfoxide, whereas the silica polyhydroxy groups enable their further modification . They still have intense luminescence, but with this method only milligrams quantities of dots might be prepared in one portion. There are three main ways of QD modification methods :
? with the use of carbodiimides as catalyst of condensation reaction between amino moiety and carboxyl group on the surface of QD
? direct connection of the proteins to the surface through the use of existing sulfur cysteine and methionine groups or histidine residues complexes by zinc ions
? by creating a self-assembled protein surfaces and non-covalent interaction .
One of the most widely studied area of QD application are spectral bar codes. Recent studies have shown that the multi-colored quantum dots are ideal fluorophores for ?multicomplexing optical coding of biomolecules.? The basis of this technique are multi-colored quantum dots precisely defined in terms of size and the number of dots enclosed in polymer capsules such as latex. The specific particles, such as peptides, proteins, oligonucleotides are covalently bound to a polymer capsules and are encoded by spectroscopic ?signature? of defined QDs . Theoretically, using only six colors and ten intensity levels one million protein or nucleic acid sequences can be encoded . This technology opens up new possibilities in the study of gene expression, screening and diagnostic tests. Another application of QDs is to track the viral cells in the body. QDs, coated for example with gold atoms, after irradiation with a specific wavelength emit light, allowing to trace the route of viral cells in a living organism . Research on quantum dots are currently being conducted in many laboratories around the world, and over the next few years should revolutionize the existing diagnostic capabilities.
Summary and outlook
Due to the increasing development of nanotechnology and its innovative potential, in 2005 the European Commission established the European Technology Platform (ETP) in Nanomedicine ? Nanotechnology for Medical Application. It aims on connecting research centers dealing with nanomedicine and set up their research to improve the quality of medical care . Through the use of specific physical and chemical properties of nanocarriers? improvement of early detection, treatment, monitoring and prevention of the diseases might be reached. According to Umberto Veronesi, a world authority in the field of oncology, most likely due to nano-solutions tumor will be just a simple disease for several years, fast to diagnose and easy to treat.
This work was supported by the European Union through the European Social Fund.
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Translation into English by the Author
Justyna SIEMIENIEC graduated from the Faculty of Chemistry, Wroclaw University of Technology. She is currently a doctoral student in the Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology. She got a scholarship of ?Enterprising Ph D. student ? investment in the innovative development of the region? (Operational Programme Human Capital), project co-financed by the European Union from the European Social Fund. Research interests: nanoparticles, synthesis of aminophosphonates and their immobilization on nanovehicles. She is the author of the chapter in the book, eight communications and posters presented at national and international conferences.
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