Current Pharmaceutical Biotechnology, 2005, 6, 17-33 17 Nanotechnology on Duty in Medical Applications

T. Kubik1,*, K. Bogunia-Kubik2 and M. Sugisaka3

1Institute of Cybernetics Engineering, Wroclaw University of Technology, Janiszewskiego 11/17, 50-372, Wroclaw, Poland; 2L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland and 3Faculty of Engineering, Oita University, Dannoharu 700, 870-1192 Oita, Japan

Abstract: At the beginning of 21st century, fifty years after discovery of deoxyribonucleic acid (DNA) double helix structure, scientific world is faced with a great progress in many disciplines of biological research, especially in the field of molecular biology and operating on nucleid acid molecules. Many molecular biology techniques have been implemented successfully in biology, biotechnology, medical science, diagnostics, and many more. The introduction of polymerase chain reaction (PCR) resulted in improving old and designing new laboratory devices for PCR amplification and analysis of amplified DNA fragments. In parallel to these efforts, the nature of DNA molecules and their construction have attracted many researchers. In addition, some studies concerning mimicking living systems, as well as developing and constructing artificial nanodevices, such as biomolecular sensors and artificial cells, have been conducted. This review is focused on the potential of nanotechnology in health care and medicine, including the development of nanoparticles for diagnostic and screening purposes, the manufacture of unique drug delivery systems, antisense and gene therapy applications and the enablement of tissue engineering, including the future of nanorobot construction. Key Words: Nanotechnology, nanoparticles, diagnostics, medicine, drug delivery.

INTRODUCTION: 50 YEARS OF DNA DISCOVERY aspects of DNA diagnostics have been noticed. These ranging from PCR-based techniques, DNA microarrays and Year 2003 marks the 50th anniversary of the discovery of microfluidics to fluorescent in situ hybridisation (FISH), the double helix structure of the deoxyribonucleic acid single nucleotide polymorphism (SNP) or short tandem (DNA) molecule. The double helix structure of DNA was repeat (STR) detection, microelectrochemical systems first described by biologist James Watson (who in 1951, then (MEMS) and MEMS-based microfluidics (lab-on-a-chip) 23 years old, travelled from the USA to work with Francis technology for DNA analysis, and many others (reviewed by Crick) and Francis Crick, an English physicist at the Demidov [4]). In parallel, ribonucleic acid (RNA)-based University of Cambridge. This discovery was the result of approaches have been developed allowing an in vitro gene work by James Watson and Francis Crick at the Cavendish expression analysis due to the use of reverse transcriptase Laboratory at the University of Cambridge, and also Maurice catalysing the synthesis of copy cDNA. Wilkins and Rosalind Franklin at King’s College, London. They found that DNA is composed of two anti-parallel More recently, introduction of DNA analogues detection strands, which wind about a common axis to form a double systems, have attracted considerable attention. These helix, and two DNA strands are held together by weak bonds systems are based on sequence-specific base paring between between the basis of each strand, forming base pairs (bp): A- the DNA strands according to the Watson-Crick model. T pairs and G-C pairs. The DNA structure model and (A-T; They include, for example, the use of peptide nucleic acid G-C) complementarity rules were published on 25th of April (PNA) technology [5], or a new class of bicyclic high 1953 in the British journal Nature [1]. This revolutionary affinity DNA analogs called locked nucleic acid (LNA) [6]. discovery of DNA led to Watson and Crick being awarded the Nobel Prize in Medicine in 1962. It is also worthy to mention the applications of triple- stranded nucleic acid structures [7] and cell-free DNA and RNA in plasma as a new tools for molecular diagnostics [8]. DNA Based Diagnostic Procedures Starting from the DNA double helix discovery 50 years All these approaches are the milestones in the development of RNA/DNA diagnostics. In the 21st century ago, through the discovery and isolation of DNA polymerase they will play an important role in many areas of diagnostics in late sixties (1958-1959), description of polymerase chain reaction (PCR) over 25 years later [2, 3], discovery of and science, including the health care, medicine or pharmaceutical industry. This is especially true, since their restriction enzymes, hybridisation, and sequencing progress is supported by nanotechnology solutions, which techniques leading to the human genom sequencing, a great have been already successfully introduced. In addition the progress in the development of DNA technology and various knowledge and understanding of the mechanisms of biological processes, such as gene expression and protein *Address correspondence to this author at the Institute of Cybernetics production (DNA synthesis, transcription into messenger Engineering, Wroclaw University of Technology, Janiszewskiego 11/17, 50- mRNA and translation into protein) help not only in the 372 Wroclaw, Poland; Tel: +48 71 3202745, +48 71 3212677; Fax: +48 71 3203408; E-mail: [email protected] development of medical diagnostic procedures but may be

1389-2010/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd. 18 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al. also applied in medical treatment, by introducing gene and [12]. Nanotechnology (in other words molecular antisense therapy. nanotechnology, molecular manufacturing) also comprises In this review we will present the recent achievements of the building of nanostructures and manufacturing nanometre- nanoscience giving some examples of their potential scale objects. Therefore, the manipulation of nanoscale applications in the fields of molecular diagnostics, particles, different kinds of microscopy, the manipulation of development of drug delivery systems, and nucleic acid molecules, constructing nanometre resolution diagnostic and based therapies. analytical devices, and nanomachines and nanorobots, can all be described by this term - nanotechnology. ANALYSIS AND OPERATIONS WITH THE USE APPLICATION IN DIAGNOSTICS NANOTECHNOLOGY; MACHINES, MATERIALS BASED ON DNA MOLECULES Biosensors The term nanotechnology is derived from the Greek Biosensors are chemical sensors, in which recognition word ‘nano’, meaning ‘dwarf’, and applies to the principles processes rely on biochemical mechanisms utilisation. They of engineering and manufacturing at a molecular level. The consist of a biological element (responsible for sampling), most common definition of nanotechnology is that of and a physical element (often called transducer, transmitting manipulation, observation and measurement at a scale of less sampling results for further processing), Fig. (1) [13-16]. than 100 nanometres. Nanotechnology is intrinsically multidisciplinary, reliant on the basic science, analytical The biological element of a biosensor contains a bio- techniques and methodologies of a number of disciplines sensitive layer, which can either contain bioreceptors or be including: chemistry, physics, electrical engineering, made of bioreceptors covalently attached to the transducer. material science and molecular biology. Nanotechnology is Bioreceptors are responsible for binding the analyte of often represented by two fundamentally different interest to the sensor for the measurement. They might be approaches: ‘top-down’ and ‘bottom-up’. ‘Top-down’ refers biological molecular species (such as antibodies, enzymes, to making nanoscale structures by machining, templating and proteins or nucleic acids) or living biological systems (e.g., lithographic techniques, whereas ‘bottom-up’, or molecular cells, tissues, or whole organisms). The type of biological element defines biological specificity conferring mechanism nanotechnology, applies to building organic and inorganic materials into defined structures, atom-by-atom or molecule- used. by-molecule, often by self-assembly or self-organisation. The physical element translate information from the Biologists/chemists are involved in the synthesis of biological element into a chemical or physical output signal inorganic, organic and hybrid nanomaterials for the use in with a define sensitivity. Thus in the biosensor the chemical nanodevices, the development of novel nanoanalytical information, ranging from the concentration of a specific techniques, and the manipulation of biological molecules sample component to a total composition analysis, is such as DNA and the evolution of molecular machines. transformed into an analytically useful signal. Thus nanotechnology includes a wide range of different Biosensors may be classified according to the type of scientific fields, from lithography (with line widths less than biological element, or to the mode of signal transduction or, one micron) to nanomachines and nanorobots. The term alternatively, a combination of the two. Concerning the nanotechnology can be understood as altering individual biological specificity conferring mechanism used one can atoms and molecules at a precise location, either chemically distinguish 5 major categories of biosensors (see Fig. (2)): or physically. Nanotechnology also seeks to develop devices 1) antibody/antigen based, that can scan and to manipulate objects at near atomic scale, such as the atomic force microscope [9], the scanning tunnel 2) enzymes based (mono- or multi enzyme systems) [17], microscope [10], the laser force microscope, laser tweezers 3) nucleic acids based (DNA, cDNA, RNA) [18], [11], or liquid chromatography - mass spectrometry devices

Fig. (1). Conceptual diagram of biosensor. A biosensor can be defined as a device that consists of a biological element (bioreceptor), and a physical element (transducer). The interaction of the analyte with the bioreceptor is designed to produce an effect measured by the transducer, which converts the information into measurable values, for example electrical signals. Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 19

Fig. (2). Different examples of bioreceptors (recognition systems) immobilized on chip surface. A – transmembrane (nanopore) sensor; B – biocatalytic (enzyme) sensor; C, D, E, – DNA based sensors, bioaffinity sensors with different mechanisms of recognition, including: C: hybridisation with nanobead labelled oligonucleotides, D: hybridisation with fluorescent labelled oligonucleotides, and E: detection of hybridised oligonucleotides carrying a given antigen by the antigen specific antibodies; F – antibody sensors with immobilized via S-layer antibodies [25, 26]; G, H – sensors utilizing cells (cell organelles) for analysis of the processes ongoing within the cell by: G: an extracellular detection or H: intracellular detection based on injected antibody immobilised on a fibre [27].

4) based on cellular interactions (cellular structures/cells), ribonucleic acid molecules, all enzymes are proteins. Some utilisating the whole cells (micro-organisms, such as enzymes require no chemical groups other than their amino bacteria, fungi, eukaryotic cells or yeast) or cell organelles acid residues for activity. Others require an additional or particles (mitochondria, cell walls, tissue slices) [19-24], chemical component called a cofactor (one or more 5) employing biomimetic materials (e.g., synthetic inorganic ions, or a more complex organic or metalloorganic bioreceptors). molecule called a coenzyme). The catalytic activity of enzymes depends upon the integrity of their native protein Biosensors with antibody/antigen as bioreceptors rely on conformation. If an enzyme is denatured, dissociated into its binding of an antigen, Ag, to a specific antibody, Ab. Forma- subunits, or broken down into its component amino acids, its tion of such Ab-Ag complexes has to be detected under catalytic activity is destroyed. Enzyme-coupled receptors can conditions where non-specific interactions are minimized. also be used to modify the recognition mechanisms. For In enzymes-based biosensors bioreceptors are made of instance, the activity of an enzyme can be modulated when a enzymes. The detection process relies on catalytic activity ligand binds at the receptor. and/or binding capabilities of these bioreceptors. In Biosensors based on nucleic acid interactions of biolo- biocatalytic recognition mechanisms, the detection is gical element are called DNA biosensors, or genosensors, or amplified by a reaction catalysed by macromolecules called biodetectors. They are used to identify small concentrations biocatalysts. With the exception of a small group of catalytic of DNA (microorganism like virus or bacteria) in a large 20 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al. sample. This relies on comparing sample DNA with DNA of array of non-nucleic acid targets with high affinity and known organism (DNA probe). Since the sample solution specificity was associated with the development of an in may contain only a small number of molecules, for proper vitro selection and amplification technique, named SELEX – analysis multiple copies of the sample DNA need to be systemic evolution of ligands by exponential enrichment, in created. For this purpose Micro-Electro-Mechanical Systems 1990 [33-35]. O’Sulllivan [36] in her review article presents (MEMS) devices are employed able to perform polymerase and discusses the potential applications of aptamers, also in chain reaction. Such a lab-on-chip system contains channels, biosensing, highlighting their advantages over antibodies for valves and chambers. analysis. Aptasensors were, for example, used for detection Biosensors based on genetically engineered bacteria are of nM concentration of thrombin in nL of an addressed volume in 10-15 min [37, 38]. Another kind of synthetic examples of biosensors with a whole cell as a biological element. They are able to detect global parameter such as biosensor is DNA biosensor based on synthetic DNA stress conditions, toxicity or DNA damaging agents (e.g. analogue - peptide nucleic acid (PNA) recognition system, heat shock, mutagenic agents) and also specific organic (e.g., referred also as PNA biosensors [39]. Due to the structure similarity of PNA and DNA, PNA is able to bind to its m-xylene and benzene derivates, naphthalene, hydrocarbons and inorganic (e.g., cadmium, arsenic, chromate, mercury, complementary nucleic acid sequence obeying the Watson- aluminium, zinc, iron) compounds as described in more Crick base-pairing rules. Interestingly, the binding affinity and specificity of complementary PNA-DNA molecules is details by Köhler et al. [20]. Cellular biosensors can be used higher as compared to traditional oligonucleotides, and is for testing and monitoring the effectiveness of drugs and therapies [28, 29]. Moreover, the microbial biosensor can independent of the ironic strength and salt concentration in monitor microbiologically influenced corrosion of metallic the hybridisation solution. In these terms interesting is also materials [21]. development of semisynthetic DNA-protein conjugates [40, 41]. Hansen and Sørensen in their minireview [23] discuss the use of whole-cell bacterial biosensors for detection and The classification of biosensors according to the quantification of various compounds and other conditions techniques used in signal transduction can be done as affecting bacterial expression of different genes. They follows [13, 42]: distinguish three types of biosensors (nonspecific, stress- 1) optical-detection biosensors (e.g., sensors based on induced, and specific biosensors) and present corresponding luminescence, absorption, polarization, fluorescence, constructs together with mechanisms these biosensors rely etc. measures); on. 2) electrochemical biosensors (e.g., conductometric, In order for bacterial cell to function as a “microbial amperometric, potentiometric, ion-sensitive); bioreporter” or a “microbial biosensor”, it has to contain two 3) biosensors based on mass-sensitive measurement (e.g., linked genetic elements (a sensing element and a reporter). surface acoustic wave, microbalance, resonant); The reporter element is always one of a typical set of genes or groups of genes, coding for proteins with an easily 4) thermal-detection biosensors. detectable presence or activity, while the sensing element in Optical-detection biosensors– this kind of biosensors most cases is a promoter for a gene or a group of genes offers the largest number of possible subcategories of all normally activated in response to a specific or general classes mentioned above. This is due to the fact that optical environmental change. Using this concept, a promoter biosensors can be used for many different types of sequence from one bacterial species can be genetically fused spectroscopy (luminescence, absorption, polarization, to a reported gene from a second species. Then the fused fluorescence, etc.) with different spectrochemical properties promoter-reporter is introduced into the host cell either as a recorded (amplitude, energy, polarization, decay time and/or plasmid or integrated into the bacterial chromosome. Dye- phase). The most commonly measured parameter of the tagged nanoparticles may be also inserted into living cells to electromagnetic spectrum is amplitude since it can be monitor metabolism or disease condition [30]. These mole- correlated with the concentration of the analyte of interest. cules consist of sensor molecules entrapped in a chemically Another parameter, energy, can reflect changes in the local inert matrix by a microemulsion polymerisation process that environment surrounding the analyte. Changes in a light produces spherical sensors in a size of 20-200 nm. polarisation can be used in order to detect whether molecules Another example is a real-time spore-based biosensor binding on some surface appeared. Fluorescence can also be system. In this system, dormant spores respond to presence used to gain information about molecular interactions since of neighbouring bacterial cells (the analyte) by producing these decay times are very dependent upon the excited state fluorescent light signals [31]. The system was used to of the molecules and their local molecular environment. identify bacterially contaminated platelets in real-time [32]. Electrochemical biosensors – all of them are designed to One of the most intriguing proposals of synthetic measure electrical values of electrochemical reactions. biosensors is an aptasensor. Aptasensors are biosensors Amperometric biosensors are based on the measurement utilizing aptamers as biocomponent, where aptamers are of the current resulting from the electrochemical oxidation or artificial nucleic acid ligands that can be generated against reduction of an electroactive species. The resulting current is amino acids, drugs, proteins and other molecules. The term directly correlated to the bulk concentration of the aptamer is derived from the Latin word ‘aptus’, meaning ‘to electroactive species or its production or consumption rate fit’, The discovery of nucleic acid sequences that bind a wide within the adjacent biocatalytic layer. Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 21

Potentiometric biosensors involve determination of the construction. In example DNA-nanopores made of a - potential difference between either an indicator and a haemolysin protein channel mounted in a lipid bilayer with reference electrode, or two reference electrodes separated by ~2 nm in diameter (Fig. (2A)) are made of organic material. a permselective membrane, when there is no significant These nanopores are able to discriminate between individual current flowing between them. The transducer may be an DNA strands up to 30 nucleotides in length, differing by a ion-selective electrode (ISE) which is an electrochemical single base substitution. There are several articles concerning sensor based on thin films or selective membranes as this subject, [52-57], while a comprehensive review is recognition elements. The most common potentiometric provided by Nakane et al. [57]. They discuss native a - devices are pH electrodes, several other ion (F-, I-, CN-, haemolysin sensor applications and present sensors based on Na+, K+, Ca++, NH4+) or gas selective electrodes (CO2, modified a -haemolysin (together with description of other NH3). Voltammetry is used in implementation of glucose organic pores and synthetic nanopores, supporting structures biosensors [43-45]. and applications). Conductometric biosensors employ ion conductometry or Safety measures. Most of the nanoparticles are impedimetic devices using integrated electrodes for considered to be safe. But it is worthy to notice, that it was monitoring of many enzymatic reactions and biological not clarified yet, if all of them are not dangerous in terms of membrane receptors. Very recently a conductometric sensor human health. As it was investigated, much in this matter for biosecurity has been reported (consisting of an immuno- depends on the size of a nanoparticle involved. Micrometre- sensor based on electrochemical sandwich immunoassay and sized clumps of nanoparticles, for example, are relatively a reader for signal measurement) for detecting foodborn inreactive because their surface area is smaller than that of pathogens [46]. the same number of individual nanoparticles, and they are too large to enter the bloodstream when breathed in. But Ion-sensitive measurement – the principle of this individual nanoparticles can pass from lungs into the measurement mode relies on the fact that when analyte ions bloodstream, and are more reactive. Toxicologist Günter interact with semiconductor the electrical potential of an Oberdörster reported that when his lab exposed rats to air electrode surface changes what can be sufficiently measured. containing 20 nm diameter polytetrafluoroethylene particles Biosensors based on mass-sensitive measurement – they for 15 minutes, most of the animals died within 4 hours. By are used for small changes in a mass of crystals due to contrast, those exposed to air with particles 130 nm in binding of chemicals detection. The changes are measured diameter suffered no ill effects. Most researchers cautioned indirectly by measuring crystals oscillation frequency. that such preliminary toxicity studies don’t warrant drastic actions, such as regulating nanoparticles. Nevertheless Thermal detection biosensors – these are micro-devices concerns about nanoparticles’ toxicity must be addressed that measure a heat of enzyme and analyte reaction. while the field is still young and exposures limited [94, 95]. Nanoparticles as Biosensors Components Biosensoring Systems There are several kinds of nanoparticles that can be used Biosensors discussed in the previous sections are devices as biosensors components. Most of them work as probes that consist of nanoscale elements. But in most cases these recognizing and differentiating an analyte of interest for elements need specialized hardware devices of larger scale diagnostic and screening purposes. In such applications for excitation and measurement [96]. The biosensors with biological molecular species are attached to the nanoparticles such hardware devices form biosensoring systems, which through a proprietary modification procedure. The probes are may vary in size significantly. The progress in the used then to bind and signal the presence of a target in a biosensoring systems development domain can be observed in sample by their colour, mass, or other physical properties. scaling down tendency. The molecular binding is a subject of the biological surface science [47], which is closely related to the research on One of the most commonly used devices in molecular modification of nanostructures properties by controlling their biology is electrophoresis apparatus. This apparatus allows structure and their surface at a nanoscale level [48, 49]. Both identification of the products of PCR amplification. It is built fields cover broad area, in which one can locate the use of on the principle that in a microenvironment with 5

Table 1. Nanoparticles for Diagnostic and Screening Purposes

Nanomaterial Characteristics, properties Applications (examples)

Multicolour optical coding for biological assays [59].

Inorganic crystals of CdSe (cadmium selenide 200-10000 atoms wide), coated Labelling of the breast cancer marker HeR2 on the with ZnS (zinc sulphide). They emit fluorescent light when irradiated with low- surface of fixed and live cancer cells (quantum dots energy light. The size of the dots (< 10 nm) determines the frequency of light emitted linked to immunoglobulin IgG and streptavidin). (i.e. colour). The dots usually have a polymer coating with multivalent bio-conjugate Nanocrystals attached, or are embedded into microbeads. Collection of dots of different size Stain actin and microtubule fibbers in the quantum dots embedded to a given microbead emits distinct spectrum of colours - spectral bar cytoplasm. code specific for this bead. Detection technique with the use of 10 intensity levels Detection of nuclear antigens in side nucleus [60]. and 6 colours could theoretically provide 106 distinct codes. Quantum dots, for example CdSe-ZnS nanocrystals, do no emit in the near infrared, so they cannot be Immunohistochemical analysis of paraffin- used for analysis in blood [58]. embedded tissue sections (fluorescent nanocrystals conjugated to antibodies) [61].

In vivo targeting [62].

SNP mapping. Freestanding, cylindrical nanoparticles with specific patterns of submicron stripes of noble metal ions, produced by alternating electrochemical reduction of the Coding in multiplexed assays for proteomics, appropriate metals. They are between 12 nm and 15 µm in width and 1–50 µm in population diagnostics and in point-of-care hand- nanobarcodes length. The striping patterns make them distinctive (like conventional barcodes) held devices. under light, or fluorescent microscopy, or mass spectrometry. Nanobarcodes are Proteins detection by either mass spectrometry or easy to make in a nearly unlimited number of uniquely identified ‘flavors’ (from fluorescence measure (after proteins immobilization 4 different metals and 8 stripes 32 896 distinct nanoparticles can be prepared) [63, 64]. on a metal surface).

Nanoparticles made of noble metals (gold or silver-enhanced gold) with Cancer diagnosis [67]. diameters between 15 to 60 nm [42, 65-67]. metallic nanobeads DNA detection assay [66]. They can be detected by the transmissive and reflective light measure, plasmon resonance, quartz crystal microbalance, and differential pulse voltametry. DNA diagnostics [68].

Efficient nucleic acid hybridisation Synthesized using standard water-in-oil microemulsion method (oligonucleotide-modified nanoparticles). (60nm in diameter). Detection of nanomolar range target DNA silica nanoparticles They were silanised, and coated by oligonucleotide before use probes (DNA conjugated nanoparticles). (DNA immobilization). They are observable by fluorescence measurements methods [69]. Ultrasmall nano-biosensors for trace analysis [69].

Ferrofluid (25-100 nm in radius) particles consist of a magnetic core surrounded by a polymeric layer (biological substrate) coated with affinity molecules, such as Imaging specific molecular targets using antibodies, for very sensitive cells or other tissue samples capture or separation. nanoparticles as magnetic resonance (MR) ferrofluid magnetic contrast agents [71]. nanoparticles, Macro magnetic beads are bigger in size. Isolation of cells (e.g. cancer of bacterial) and many magnetic beads They might be coated with streptavidin to bind to biotin with a single-stranded important biological molecules (e.g., DNA, DNA probes specific for a bioagent or sample DNA attached. They are detectable peptides) from complex mixtures, including blood, trough amperometry or resistance measure. They can be also manipulated in a tissues and bodily fluids. magnetic field [70].

Carbon cylinders rising in the process of folding graphitic layers. The cylinders have remarkable strength and unique electrical properties making them insulting, Promotion of electron transfer reactions when semiconducting or conducting depending on their structure. They may be used to fabricate electrodes for the oxidation of composed of a single shell – single-walled nanotubes (SWNTs), or of several biomolecules including dopamine, protein and b- shells – multi-walled nanotubes (MWNTs) [72, 73]. They are produced by carbon nanotubes nicotinamide adenine dinucleotide [77-80]. microwave plasma enhanced chemical vapour deposition. Deposition’s parameters: pressure, substrate temperature, microwave power and gas flow ratio Extracellular analysis (glucose biosensor constructed by immobilization of enzyme on of CH4/ (CH4+H2) have influence on cylinders structure. Carbon nanotubes may be grown on a wide variety of substrates (including quartz and glass slides, multi-walled carbon nanotubes [81, 82]). platinum substrates) in the presence of catalyst (like nickel). Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 23

(Table 1) contd….

Nanomaterial Characteristics, properties Applications (examples)

Average length of these nanoparticles is of 25 mm order. Mean diameter may vary from 30 nm up to 150 nm. But with the use of super-lattice nanowire pattern transfer individual semiconductor nanowires can be created that are as little as 8 nm in diameter with the same distance between each wire (Jim Heath, California in vivo diagnostics (integration of vertically Institute of Technology, Pasadena) [74]. aligned carbon nanofibre with either absorbed or covalently-linked plasmid with the intracellular In most cases electrical properties of carbon nanotubes are utilized for domains of viable cells for controlled measurement purposes. When used in biosensing, carbon nanotubes have specific biochemical manipulation [83]). biomolecules attached.

There are some other materials (no carbon), which possess ability to perform the conformation of nanorings and nanotubes [75, 76].

In situ measurements of benzopyrene tetrol in Optical fibres pulled down to tips having distal end sizes of approximately 30–60 single cells with the antibody-based nanoprobe nm. Fabrication procedure involves pulling from a larger silica optical fibre using (small size of optical fibres allows sensing a special fibre-pulling device what yields fibre with submicron diameters. One intracellular/intercellular physiological and end of such fibre is polished from 600-mm silica/silica to a 0.3 mm finish. The biological parameters in microenvironments) other end is pulled then to a submicron length using a fibre puller. Thus the distal [14, 27]. end of the fibre reaches 60 nm size. To prevent light leakage of the excitation optical fibres Specific detection of target DNA at zeptomole light on the tapered side of the fibre, the side wall of the tapered end can be levels (in 10 µl of a 100 attomolar solution), in coated with a thin layer of silver, aluminium or gold (100–300 nm) leaving the the presence of non-cDNA [86]. distal end of the fibre free. These nanoparticles are not subject to electromagnetic interferences from static electricity, strong magnetic fields, or surface potentials. Analysis of mRNA isoforms in human cancer Fibreoptic nanoprobes can be covalently bound either with bioreceptors, such as cell lines in conjunction with a new enzymatic antibodies, or with other, synthetic receptors, such as cyclodextrins [84, 85]. detection method termed RNA-based annealing, selection and ligation (RASL) [87].

Molecular-scale pores fabricated from variety solid-state materials (like silicon nitride) by ion-beam sculpting technique [88, 89].

The technique uses low-energy ion beams to slowly shape the surface of a Manipulation and electronically material, while a feedback loop enables single-nanometre control over the pore registering of single DNA molecules dimension. Nanopores can be fabricated in two ways: they can be created from a in aqueous solution [91, 92]. cavity in the membrane under conditions where the sputtering erosion process Discrimination and characteristic of unlabelled nanopores dominates, or can be made by filling in larger pores under conditions where the DNA molecules at low copy number [52]. lateral mass transport process dominates. It has been suggested that the nucleic acid The depth of nanopore fabricated in a membrane 5–10 nm thick is smaller than sequences could be determined at rates exciding the molecule persistence length (50 nm for dsDNA), and a pore diameter 3-nm is 1000 bases per second [93]. slightly larger than the cross-sectional size of the molecule (~2 nm). In another attempt nanopores are created as an array of cylindrical gold nanotubes with as small as 1.6 nm inner diameter [90]. of 10 microlitres using 30 PCR cycles for about 1.5 hours fixed locations (spots). The spots can be either printed on the (i.e., PTC-200 DNA Engine Line Gradient Feature, MJ microarrays by a robot, or synthesized by photolithography Research Inc., Waltham MA, USA). Currently, even more or by ink-jet printing. Each spot contains a number of precise devices are available. identical DNA fragments of lengths varied from 20 to Other most productive technological solutions in hundreds of nucleotides. According to quick napkin calculations, the number of DNA molecules in a microarray biosensing are DNA microarrays. In fact, there are several spot is 107- 108. Typically array size is ~25x25 mm, the names for this technology - DNA microarrays, DNA arrays, diameter of spot is ~0.1 mm. Microarrays for genotypic DNA chips, gene chips, and others. But all of them refer to analysis can be employed for both diagnostic purposes and the technology that makes use of DNA microarrays (please assessment of drug responses [99]. There are different ways note the recent review of Howbrook et al. describing the how microarrays can be used to measure the gene expression developments in microarray technologies [98]). levels. In general the measuring starts with the mRNA Microarrays use the preferential binding of comple- extraction from the cells, followed by fluorescent labelling. mentary single-stranded nucleic acid sequences. They are The labelling is usually done during the synthesis of usually glass slides on which DNA molecules are attached at complementary to extracted RNA single-stranded copy DNA 24 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al.

(cDNA) fragments. Then labelled cDNA samples are washed an example, a high-throughput biochemical microprocessor over the microarray and the amount of nucleic acid bound to is able to perform rapid and parallel DNA sequencing on 96 each spot is measured by a level of (excited by a laser) samples to 500 bases, corresponding to a throughput of ~100 fluorescence emitted. This gives the raw data (the hybridised kilobases per hour. An integrated device for PCR ampli- microarray images) that must be additionally analysed by the fication and electrophoretic analysis was also interesting. image analysis software. The final data are produced after This was a fully integrated system in glass for the manipu- some scaling procedures applied to the obtained data. lation, amplification, and capillary electrophoresis separation of submicrolitre volumes of DNA [110]. In this system, the use of a low-volume reactor with 280 nanolitre PCR chambers and thin-film heaters permitted thermal cycle times as fast as 30 sec. The amplified product was labelled with an intercalating fluorescent dye and injected directly into a microfabricated capillary electrophoresis system. The reac- tion could start with as few as 20 DNA template copies/ microlitre (5-6 per chamber). During the same conference, miniaturized high-speed thermal cycling chambers were shown. These chambers were able to complete 10 PCR cycles in 30 seconds and successfully amplify a DNA sample in fewer than 4 minutes. Moreover, a method for the multiplex detection of polymorphic sites and direct determination of haplotypes in 10-kb DNA fragments, using single-walled carbon nanotube atomic force microscopy probes, was also described [111]. This technique was based on the hybridisation of labelled oligonucleotides to complementary target DNA sequences. It allowed the direct determination of haplotypes in patient samples and had been employed for study on UGT1A7, a cancer risk-factor gene. In addition, a novel method for electrochemical detection of DNA hybridisation with the use of conductive hydrogels was proposed. These micro- and nanotechnological tools are being used to sequence genomes and diagnose diseases. The analysers mentioned above may allow much faster, more specific, and more precise DNA analysis and DNA-based diagnostic procedures than used presently. The reduction of sample volume, like the amount of necessary DNA template, is an additional advantage, especially in the case of a limited availability of samples (e.g., for pediatric patients). Employ- ing more efficient machines may also improve and shorten the time of analyses, which can be of particular importance. Biosensors that include transducers based on integrated circuit microchips are often referred as biochips. In general, Fig. (3). Schemes of different nanoparticles used for diagnostic biochip consists of an array of individual biosensors that can and screening purposes. be individually monitored and generally are used for the analysis of multiple analytes. Lab-on-chip technology One of the first microdevices to both amplify and detect involves micrototal analysis systems that are distinguished PCR products without intermediate steps was reported in from simple sensors by their ability to conduct a complete Wooley et al. [100]. This device, composed of a silicon analysis [112]. The recent advances in biochip/biosensor reaction chamber attached to a glass capillary electrophoresis technology in Central Europe were reported by Gauglitz separation channel, had a 10-microlitre volume and [113] in his meeting report. performed amplifications in 15 min. Since then, a great number of different microfluidic PCR devices have been NANOPARTICLES AS CARRIERS OF THERAPEU- fabricated in silicon [101-104], glass [105, 106], silicon- TIC MOLECULES glass hybrids [107], and in fused silica capillaries [108]. From nanotechnology there is only one step to nanome- During the BioMENS and Biomedical Nanotechnology dicine, which may be defined as the monitoring, repair, World 2000 conference (reviewed by Wooley [109]) many construction, and control of human biological systems at the interesting machines for biochemical analysis were molecular level, using engineered nanodevices and nano- presented. These included mainly micromachines allowing structures. It can also be regarded as another implementation different DNA analyses to be performed, e.g., PCR ampli- of nanotechnology in the field of medical science and fication, electrophoresis, and sequencing. Here, to serve as diagnostics. One of the most important issues is the proper Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 25 distribution of drugs and other therapeutic agents within the Nanoparticles here are defined as being submicronic (< 1 patient’s body. mm) colloidal systems generally made of polymers Drug delivery. Targeting the delivery of drugs to (biodegradable or not). They were first developed in the diseased lesions is one of the important aspects of the drug middle 1970s by Birrenbach and Speiser [117]. Nano- delivery systems. To convey a sufficient dose of drug to the particles generally vary in size from 10 to 1000 nm. lesion, suitable carriers of drugs are needed. Nano- and Depending upon the process used for the preparation of microparticle carriers have important potential applications nanoparticles [118-120], nanospheres or nanocapsules can be for the administration of therapeutic molecules. Liposomes, obtained. The drug is dissolved, entrapped, encapsulated, or Fig. (4), have been used as potential drug carriers instead of attached to a nanoparticle matrix. conventional dosage forms because of their unique Nanocapsules are vesicular, reservoir systems in which advantages, which include the ability to protect drugs from the drug is confined to a cavity (an oil or aqueous core) degradation, target the drug to the site of action, and reduce surrounded by a unique thin polymeric membrane. the toxicity of side effects [114]. However, developmental Nanospheres are polymeric matrix systems in which the drug work on liposomes has been limited, due to inherent is physically and uniformly dispersed throughout the problems such as low encapsulation efficiency, rapid leakage particles. of water-soluble drugs in the presence of blood components, and poor storage stability. Thus most of the early clinical Polymers suitable for preparing nanoparticles include: poly(alkylcyanocrystalates), poly(mythylidene malnolate studies with liposomes met with failure because the injected vesicles were rapidly removed by phagocytic cells of the 2.1.2), polyesters, e.g., poly(lactic acid), poly(e caprolac- immune system. This obstacle has now been overcome with tone), and their copolymers. For the nanosphere preparation the development of so called ‘stealth liposomes’ that contain natural macromolecules, such as proteins and polysaccha- rides, non-polar lipids, and metal oxides and silica, can also an outer coating of a synthetic polymer that protects the be used. liposomes from immune destruction. In some cases, nanoparticles are more efficient drug carriers than liposomes In recent years, biodegradable polymeric nanoparticles due to their better stability [115] and possess more useful have attracted considerable attention as potential drug control release properties. These are the reasons why many delivery devices with the prospects of their applications in drugs have been associated with nanoparticles (e.g., controlling drug release, their ability to target particular antibiotics, antiviral and antiparasitic drugs, cytostatics, organs/tissue, as carriers of oligonucleotides in antisense vitamins, protein and peptides, including enzymes and therapy, DNA in gene therapy, and in their ability to deliver hormones (please note the references for this paragraph of proteins, peptides and genes through oral administration the paper)). [121]. The use of colloidal, particulate carrier systems (25 Drug delivery systems can be classified according to (1) nm to 1 mm in diameter) for drug delivery is discussed by their physical form or (2) their functional properties. In the Barratt in his review [122]. Other applications of nano- latter case three groups have been proposed, called first-, particulate systems will be discussed below in more detail. second- and third generation systems [116]. Encapsulation is an attractive delivery option for a variety of drugs. It can reduce systemic toxicity, protect First generation systems. This group includes micro- vulnerable molecules from degradation in the digestive tract, capsules and microspheres for control chemoemobilisation provide controlled release properties or mask an unpleasant and control release of proteins and peptides or for drug taste. The resulting capsules have a wall thickness of 10-40 delivery within the brain. Although they are capable of nm and rage from 20 nm to 20 mm in diameter, with an delivering the active substances specifically to the intended target, they have to be implanted as closely as possible to the exact size controlled via the production process [123]. site of action, and therefore they cannot be considered as The use of nanocapsules as drug carriers is associated ‘carriers’. with a number of advantages. For example, poly(alkylcy- anocrystalate) nanocapsules were shown to protect insulin Second generation systems. To this group belong so- from degradation by digestive enzymes and to pass called true carries: liposomes, nanocapsules, and in vitro across the interstinal mucosa [124]. It was also reported that nanospheres (called passive colloidal carriers), and certain (in rat) encapsulation of somatostatin analogue within active carries which release their contents after a specific nanocapsules given by oral route improved and prolonged its signal, such as temperature-sensitive liposomes and therapeutic effect [125] while encapsulation in chitosan magnetic nanospheres. They are less than 1 mm in diameter nanoparticles improved a nasal absorption of insulin [126]. and are capable of releasing an active product at the intended Moreover, encapsulation provides effective protection of the target carrying it there after administration by a general gastrointestinal mucosa, which was shown e.g., by reducing route. Their usage is limited as they are rapidly removed the side-effects of diclofenac [127] encapsulated in poly from the circulation by phagocytic cells and cannot cross (lactic acid) nanocapsules and also by reducing drug-related normal capillary endotherium. irradiation, e.g., after administration in the intramuscular Third generation systems. The third generation systems route [128]. are also true carries based on monoclonal antibodies, which characterise with a capability of specific recognition. To this To make drug carries ‘invisible’ to macrophages and thus to reduce their uptake by phagocytic cells a special strategy group belong monoclonal antibodies per se and liposomes, has been applied for preparation of matrix-structured nanoparticles (nanocapsules and nanospheres) piloted by monoclonal antibodies or their ligands. nanospheres. This technique is based on the nanoparticle 26 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al.

Fig. (4). Principles of DNA transfer based on plasmid DNA - liposome (lipoplex) gene delivery system. Neutral and cationic lipids form a bilayer liposome with hydrophobic (inside the spherical structure) and hydrophilic (outside) parts. Negatively charged plasmid DNA molecules carrying therapeutic DNA fragments are not able to enter the cells due to the electrostatic interaction with the cell membrane. DNA assembly within the central cavity of cationic liposomes results in lipoplex generation. In this form, the plasmid (as a part of a lipoplex) can enter the cells via endocytosis. After degradation of endosomes in cytoplasm, DNA molecules are released; they enter the cell nucleus, where transcription and translation of a ‘corrected’ gene take place. Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 27 surface modification, e.g., with poly(ethylene glycol) (PEG) Antisense oligonucleotides have emerged as potential [129, 130] that provides a ‘cloud’ of hydrophilic chains at gene-specific therapeutic agents and are currently under- the nanoparticle surface repelling plasma proteins. For going evaluation in clinical trials for a variety of diseases. example, this kind of particle has been loaded with These include advanced carcinoma [136-139], non-Hodgkin’s tamoxifen for antiestrogen therapy in the treatment of lymphoma [140, 141], acute myeloid or lymphoblastic hormone-dependent tumours [131]. Nanoparticles prepared leukaemia [142], and chronic myelogenous leukaemia. from PLGA PEG co-polymers have been shown to increase (CML) [143]. the circulating half-life of cisplatine [132]. Similar Antisense oligonucleotides are molecules that are able to approaches have been applied to the reservoir-type polymer- inhibit gene expression, being therefore potentially active for based drug carrier nanocapsules with the aim of creating the treatment of viral infections or cancer. However, the long-circulating systems with a high loading capacity of problems such as the poor stability of antisense oligo- lipophilic drugs [133], e.g., in solid tumours treatment nucleotides versus nuclease activity in vitro and in vivo, and nanocapsules made of PLA PEG loaded with tetra their low intracellular penetration have limited their use in (hydroxyphenyl)chlorin were used [134]. therapeutics [144, 145]. In order to increase their stability, to Another strategy for preparing long-circulating colloidal improve cell penetration and also avoid non-specific systems can be considered as biomimetic in that it seeks to aptameric effects (leading to non-specific binding of imitate cells or pathogens, which avoid phagocytosis by antisense oligonucleotides), the use of particulate carriers reducing or inhibiting complement activation. e.g., heparin, such as liposomes or nanoparticles, has been considered. the anionic polysaccharide anticoagulant, enabled to inhibit Very recently, it was reported that antisense oligo- several steps of the complement cascades, can be used to nucleotides could be encapsulated in nanocapsules with a modify the surface of nanoparticles and provide a size of 350 + 100 nm. A formulation of these capsules might biomimetic effect [135]. have special importance for oligonucleotide delivery. The A gold nanoshel-polymer composite that combines first experiments on the treatment of RAS cells expressing infrared optical activity with the uniquely biocompatible the point-mutated Ha-ras gene were promising [119]. In properties of gold colloid can be next example of another approach, nanocapsules loaded with an aqueous core nanoparticle engineered for drug delivery. Metal nanoshells have been developed for the encapsulation of antisense are concentric sphere nanoparticles consisting of a dielectric oligonucleotides. They were shown to effectively protect (typically gold sulphide or silica) core and a metal (gold) oligonucleotides from degradation in biological fluids, e.g., shell. By varying the relative thickness of the core and shell in an experimental model of Ewing sarcoma for phospho- layers, the plasmon-derived optical resonance of gold can be trioateantisense oligonucleotides directed against EWS Fli-1 shifted in wavelength. By varying the absolute size of the chimeric RNA [146]. gold nanoshell, it can be made to either selectively absorb It is also worthy to mention recently described nucleic (for particles diameters < 75 nm) or scatter incident light. acid molecules that can find practical applications in When optically absorbing gold nanoshells are embedded in a antisense therapy, named small interfering RNA, or siRNA matrix, illuminating them at their resonance wavelength [147, 148]. siRNA is a short RNA duplex between 15 to 21 causes the nanoshells to transfer heat to their local nucleotides in length. These duplexes have two-nucleotide environment. This photothermal effect can be used to overhangs on their 3-prime ends and are phosphorylated on optically remote control drug release in a nanoshell-polymer their 5-prime ends. They can be used for, so-called, gene composite drug material [48]. silencing. Once transfected into cells, siRNA, in conjunction with cellular machinery, targets messenger RNA molecules ANTISENSE AND GENE THERAPIES containing an identical sequence for degradation in a Antisense therapy. Nucleic acids can be used not only to catalytic manner. The degraded message is no longer diagnose and monitor, but also to prevent and cure diseases, functional in translation (the biosynthesis of protein) and as they constitute the bases of antisense and gene therapies. thus in the expression of the corresponding gene. Designer Application of an antisense strategy to regulate the siRNA molecules targeting a gene of interest can be transcription of disease-related genes in vivo has an transfected into cells to suppress the expression of that gene. important therapeutic potential to treat or cure a variety of Moreover, the effects of the suppression can then be assayed diseases and abnormal physiological conditions. by a number of different biochemical, molecular and cellular biology methods to understand the role of the gene in Theoretically, an antisense oligonucleotide is a short biological model system under study. fragment (15-20 bp) of deoxynucleotides characterised by a On the other hand, also very recently, a successful ODN- sequence complementary to a portion of the targeted mRNA. based approach termed decoy ODN has used synthetic ODN The aim of the antisense strategy is to interface with gene containing an enhancer element that can penetrate cells, to expression by preventing the translation of proteins from bind to sequence-specific DNA-binding proteins and mRNA. There are a few mechanisms of mRNA inactivation interfere with transcription and . Transfection [119], including (i) sterical blocking of mRNA by antisense in vitro in vivo binding and destruction antisense-mRNA hybrids by RnaseH of cis-element double-stranded ODN (decoy ODN) results in enzyme, (ii) formation of triple helix between genomic attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements double-stranded DNA and oligonucleotides, or (iii) the cleavage of target RNA by ribozymes. with subsequent modulation of gene expression [149]. The principles of the decoy strategy and how to design decoy 28 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al.

ODN can be found in Tomita et al. report [150]. This administration of DNA vaccines. As an alternative to approach provides a new powerful tool in a new class of intramuscular administration of plasmid DNA, researchers anti-gene strategies to treat various diseases or as a research have been investigating targeting plasmid DNA to the skin tool to examine the molecular mechanisms of expression of a using intradermal needle injection, needle-free jet injection specific gene. devices, the gene gun, or recently topical delivery [160] of formulated plasmid in the form of a patch, cream, or gel. The Gene Therapy and Administration of DNA Vaccines latter method may provide many advantages in terms of cost and patient compliance [161]. Among other nanoparticles, Gene therapy is a recently introduced method for chitosan, a biodegradable polysaccharide comprise of treatment or prevention of genetic disorders by correcting primarily D-glucosamine repeating units, has been proposed defective genes responsible for disease development based by several groups as an alternative non-viral delivery system on delivery of repaired, or the replacement of incorrect, for plasmid DNA. Selective chitosan polymers and chitosan genes. The most common approach for correcting faulty oligomers have been found to efficiently condense plasmid genes is insertion of a normal gene into a nonspecific DNA and to transfect several different cell types in vitro and location within the genome to replace a nonfunctional gene. in the intestines, colon, nose, and lung [160, 162]. Chitosan An abnormal gene could be also swapped for a normal gene nanoparticles were also applied for DNA vaccination by the through homologous recombination or repaired through oral route [163]. selective reverse mutation, which returns the gene to its normal function. The principals of “ideal” synthetic non-viral vectors have been presented and discussed by Rubanyi [164]. Such a gene There is the wide range of target cells and diseases, like delivery system may incorporate many of the advantages of cancer, infectious, cardiovascular, monogenic (e.g., viruses, which during long evolution acquired the prefection hemophilias) diseases, and rheumatoid arthritis, for which of gene delivery to host organisms, such as dense DNA clinical studies are ongoing [151, 152]. In fact, the first “packing”, cell recognition, cellular uptake, cytosolic disease approved for gene therapy treatment was adenosine trafficking, efficient nuclear uptake and gene expression in deaminase (ADA) deficiency, and the first two patients were the host nucleus. From the other hand, this vector should treated with intramuscular injections of pegylated bovine lack the unwanted properties of viruses, as pathogenecity, ADA (PEG-ADA) in September 1990. Now a days, over 13 cell toxicity, immune and inflammatory reactions, etc.). In years later, Muul et al. [153] reported the results of long- this “ideal” system, after dense DNA packing is accom- term follow-up and data collected from these origial patients, plished the surface of synthetic particles (which is usually which provided novel information about the longevity of T positively charged, Fig. (3)) needs to be “shielded” (e.g., by lymphocytes in humans and persistence of gene expression PEG) so that they do not attach to blood elements or to each in vivo from vectors. In addition, one of the latest reports on other, and therefore have an extended circulating plasma gene therapy demonstrates the successful treatment of half-life (passive targeting to “leaky” vessels). For active patients with haemophilia B, with a defect in a gene targeting to selected cells/tissues, the particles should be encoding blood coagulation factor IX [154, 155] and patients engineered to contain on the surface specific ligands for cell with haemophilia A having a defect in a gene that encodes recognition, while engineering viral cell membrane fusion factor VIII [156]. In these cases, patients’ fibroblasts proteins to the particle coat should be used to facilitate the transfected with a plasmid containing sequences of the factor cell entry. This gene delivery vector should also characterise VIII gene (haemophilia A treatment) and adeno-associated with enhanced cellular trafficking, prevented intracellular viral vectors expressing human factor IX (haemophilia B) DNA degradation, nuclear uptake of DNA facilitated by viral were used for gene transfer. nuclear localisation signal peptides, augmented chromo- Application of nanotechnological tools in human gene somal localisation, and gene expression regulated by specific therapy has been reviewed widely by Davis [157]. He transcriptional control elements. In such an “ideal” system described non-viral vectors based on nanoparticles (usually nanosize particles could find practical implementation. 50-500 nm in size) that were already tested to transport Also Saxl [165, 166] in her reports discusses the plasmid DNA. He emphasized that nanotechnology in gene development of alternative vectors based on synthetic, non- therapy would be applied to replace the currently used viral viral systems. She proposed the use of the following nano- vectors by potentially less immunogenic nanosize gene vectors for gene therapy and DNA vaccines: carriers. So delivery of repaired genes, or the replacement of incorrect genes, are fields where nanoscaled objects could be 1) polymer-DNA complex vectors, in which polymer wrap introduced successfully. around the DNA forming particles that range 25-300 nm in diameter. Practical advantages of such complexes are On the other hand, genetic immunization with DNA that the polymer protects the DNA and may also vaccines has emerged as one of the most promising improve the cell transfection efficiency. applications of non-viral gene therapy [158, 159], having a number of the potential advantages over conventional 2) liposome-DNA complex vectors, which characterise vaccines. These include: (i) the high stability of plasmid with DNA condensation within a lipid bilayer DNA, (ii) low manufacturing costs, (iii) lack of infection risk (liposome). associated attenuated viral vaccines, (iv) the capacity of 3) polymer-oligonucleotide complexes. In these complexes target multiple antigens to one plasmid, and (v) the ability to peptide nucleic acid (PNA) is delivered to the cell elicit both humoral and cellular immune responses. Until nucleus and the faulty gene is reprogrammed. Their recently, intramuscular injection was the primary route of usage may be more beneficial than gene therapy. Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 29

She also highlights the possible implementation of transport peptides and proteins linked to the vitamin B12 nanobiomimetics for DNA vectors, including the develop- from the intestine into the circulation. To maximize the ment of nanoengineered polymeric materials that are bio- potential of the delivery system, the pharmaceutical is being inspired by viral/toxin strategies. incorporated within biodegradable nanoparticles, and coated In another review Lemieux summarises some of the latest with vitamin B12 [173]. This has advantages of protecting the technological advances to increase immunogenicity of DNA pharmaceutical from proteolysis within the intestine, of vaccines administered by the im. and id. routes towards the amplifying the uptake capacity of the oral delivery system, and improvement of mucosal, humoral and cellular immune of eliminating the need for conjugation of pharmaceuticals to responses mostly against cancer and infectious disease vitamin B12. There have been other proposals utilizing the pre- existing mechanisms of molecule delivery within the body, such antigens [167]. as drug targeting biodegradable nanoparticles coupled to folic Moreover, during the 2nd Annual BioMENS and acid [174]. Biomedical Nanotechnology World 2001 conference In addition to oral administration (reviewed by Berry [168]) many interesting achievements of Other applications. nanoscience for drug delivery were presented. For example, [121], the use of nanoparticles for nasal [175] and ophthal- Desai described how nanoporus interfaces that are mic delivery of drugs [176, 177] has been investigated. selectively permeable to bimolecular species could be Nanoparticles have enabled crossing the blood-brain barrier that fabricated using sacrificial lithography and that nanoporus represents an insurmountable obstacle for a large number of drugs, including antibiotics, antineoplastic agents, and a variety cylinders containing semiporus regions can be used for drug of central nervous system active drugs, especially neuropeptides delivery. During the same conference Ferrari described a [178, 179]. Furthermore, nanosize carriers of such molecules novel nanopump, an externally controlled, implantable drug as vitamins, for example vitamin A [180] and E [181], have delivery device for the long-term release of drugs, which is potential applications in dermatology and cosmetics. targeted for the use in programmable drug delivery systems [168]. It was also proposed that nanopore membranes, which act as sieves for the selective passage of molecules, could be CREATIONS OF ARTIFICIAL ORGANS AND used in capsules with cells for transplantation and in drug IMPLANTS delivery and release [168]. It is also important to mention Another field where the achievements of nanotechnology about cell-based strategies for applications in drug discovery can be practically applied is creation of artificial cells, for testing drugs before they enter expensive animal and tissues and organs. Artificial cells are being actively clinical trails. One of such a new approaches, termed high investigated for use in the replacement of defective or content screening (HCS), uses cells as biosensors, exploiting incorrectly functioning cells and organs, especially related to the sensitive and specific molecular detection and metabolic functions. The earliest routine clinical use of amplification systems that cells use to sense changes in their artificial cells is in the form of coated activated charcoal for external environment [168]. hemoperfusion. The implantation of encapsulated cells is In this view also the opinion of Ross and Ginsburg being studied for the treatment of diabetes, liver failure, should be quoted. They underlines the role of integration of kidney failure and the use of encapsulated genetically diagnostics and therapeutics which represents a major new engineered cells for gene therapy. Artificial cells are also opportunity for them to emerge as leaders of the new considered for drug delivery and for other uses in medicine, guiding the selection, dosage, route of biotechnology, chemical engineering and medicine [182, administration and multi-drug combinations, and producing 183]. increased efficacy and reduced toxicity of pharmaceutical The problems in creating the biological systems (artificial products [169, 170]. cells) are described by Pohorille and Deamer [184]. These Treatment of patients with peptide and protein authors also discuss the potential applications of artificial pharmaceuticals. Currently, this treatment is performed cell-like structures in pharmacology and medical diagnostics mostly by injection, with accompanying patient discomfort, pointing out the properties of an ideal minimal cell. increased medical costs, and reduced patient compliance. Chang and co-workers present the future prospects for Therefore the systems of delivery of peptides and proteins by artificial blood, especially of the artificial red blood cells the oral route have attracted considerable attention as being (RBC) [185-187]. Very recently, they have reported the much easier and more acceptable. Unfortunately, this route development of new artificial red blood cells that are more cannot be used with most proteins and peptides, due to both like natural RBC. Their novel nano-dimension red blood cell the degradation of these molecules within the intestine and substitute is based on ultrathin polyethylene glycol- their poor uptake across the intestinal wall. To potentially polylactic acid (PEG-PLA) membrane nanocapsules (80-150 overcome these problems, it has been shown that it is possible nm diameter) containing hemoglobin (Hb) and enzymes to utilize the uptake mechanism of vitamin B12 to enhance the [186]. It is worthy to note that blood substitutes based on oral uptake of various peptide and protein pharmaceuticals. In modified hemoglobin, polyhaemoglobins (PolyHb) and particular, molecules such as luteinizing hormone releasing perfluorochemicals, are already in advanced phase III hormone (LHRH) analogues, alpha-interferon, erythropoietin, clinical trials while conjugated haemoglobins are in phase II and granulocyte colony stimulating factor (G-CSF) have been clinical trial. And the circulation time of the novel artificial studied. These pharmaceuticals have been linked covalently to RBC of Chang et al. (containing haemoglobin and RBC the vitamin B12 molecule [171, 172]. This system relies upon enzymes with membrane formed from composite copolymer the natural uptake mechanism for vitamin B12 to co- of PEG-PLA) is double that of PolyHb [187]. 30 Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 Kubik et al.

More theoretical proposal of an artificial red blood cells to a general question: would synthesized nanomaterials be is the mechanical red blood cell, called respirocyte, designed able not only to replicate the properties of their natural by Freitas [188, 189]. This was the first detailed design study equivalents (cell membranes, tissues and bone marrow), but of a specific medical nanodevice (of the kind proposed by also prompt biological systems to build up on these Dexler in “Nanosystems”). The proposed respirocyte is materials, and to produce self-assembling structures? Studies about 1 micron in diameter and just flows along the on such nanostructures lead to promising materials with bloodstream. It is a spherical nanorobot made of 18 billion potential uses as implants and therapies. Moreover, they may atoms. The respirocyte is equipped with a variety of someday show how the cells interact with nanometer-sized chemical, thermal, and pressure sensors and an onboard objects in their own world. nanocomputer. This device is intended to function as an artificial erythrocyte, duplicating the oxygen and carbon NANOTECHNOLOGY AND NANOMEDICINE IN dioxide transport functions of red cells, mimicking the action THE FUTURE of natural hemoglobin-filled red blood cells. It is expected to be capable of delivering 236 times more oxygen per unit Medical diagnosis, proper and efficient delivery of pharmaceuticals, and development of artificial cells are the volume than a natural red cell. Specially installed equipment medical fields where nanosize materials have found practical enables this device to display many complex responses and behaviours. Additionally, it has been designed to draw power implementations. As suggested by Freitas, the application of from abundant natural serum glucose supplies, and thus is nanotechnology to medicine, nanomedicine, subsumes three capable of operating intelligently and virtually indefinitely, mutually overlapping and progressively more powerful whilst red blood cells have a natural lifespan of 4 months. molecular technologies [195]. First, nanoscale-structured Thus these artificial red blood cells are theoretically able to materials and devices that can be fabricated today hold great promise for advanced diagnostics and biosensors, targeted provide oxygen and can do it even more effectively than an erythrocyte. It could replace defective natural red cells in drug delivery and smart drugs, and immunoisolation therapies. Second, biotechnology offers the benefits of blood circulation. An onboard nanocomputer and numerous molecular medicine via genomics, proteomics, and artificial chemical and pressure sensors enable complex device engineered microbes. Third, in the longer term, molecular behaviours that are remotely reprogrammable by the machine systems and medical nanorobots will allow instant physician via externally applied acoustic signals. So far pathogen diagnosis and extermination, chromosome several other nanoscale devices have been described in the replacement and individual cell surgery , and the literature. Respirocyte is one of the proposals derive from the in vivo efficient augmentation and improvement of natural field of molecular nanotechnology and nanorobot physiological function. construction with intended practical implementation in medicine [190-193]. There are several other intriguing, still theoretical, It is also expected that new techniques will allow tissues proposals for practical applications of nanomechanical tools and organs to be grown artificially on nanopatterned into the fields of medical research and clinical practice. One scaffolds to obtain internal tissues implants. In parallel function of nanodevices in medical sciences could be the external tissue products are under the development for replacement of defective or incorrectly functioning cells, artificial skin, tissue reconstitution and enhanced wound such as the respirocyte proposed by Freitas [188, 189]. It has treatment. Moreover, through biomimickry or minerals laid also been postulated that nanomachines could distribute drugs within the patient’s body. Such nanoconstructions down as shells and exoskeletons, new bone will be encouraged to grow to heal broken bones and teeth, which could deliver medicines to particular sites, making more could find the practical applications for dental and bone adequate and precise treatment possible [196-198]. Such marrow replacement [165, 166]. devices would have a small computer, several binding sites to determine the concentration of specific molecules, and a One of the artificial nanostructures that can interact with supply of some ‘poison’ that could be released selectively. and replace natural biological materials has been proposed Similar machines equipped with specific ‘weapons’ could be by Taton and co-workers. In the report from one of the used to remove obstructions in the circulatory system or meetings of American scientists (American Chemical identify and kill cancer cells. It has been also proposed that Society ProSpectives, Berkeley, California, USA, 2001) nanorobots may be modified bacteria and viruses that Taton [194] presents a very intriguing proposal of an already have most of the motorisation and target delivery of artificial bone relying on designing the synthetic substitutes genetic information [165, 166]. of collagen. Research on designing self-assembling, Moreover, nanorobots, operating in the human body, synthetic substitutes for collagen has been conducted by a could monitor levels of different compounds and store that group of Stupp at Northwestern University, Evanston, information in internal memory. They could be used to Illinois. They proposed an artificial material, composed of rapidly examine a given tissue location, surveying its amphiphilic molecules bearing a long hydrophobic alkyl biochemistry, biomechanics, and histometric characteristics group on one end and ahydrophilic peptide on the other, in greater detail. This would help in better disease which was able to spontaneously assemble into cylindrical diagnosing [199, 200]. The use of nanodevices would give structures that resemble collagen fibrils. Moreover, these the additional benefits of reduced intrusiveness, increased cylinders guided the formation of hydroxyapatite crystallites. patient comfort, and greater fidelity of results, since the What is even more important, they formed crystallites target tissue can be examined in its active state in the actual characterized with orientations and sizes similar to those in host environment. natural bone. Taton emphasized that these observations lead Nanotechnology on Duty in Medical Applications Current Pharmaceutical Biotechnology, 2005, Vol. 6, No. 1 31

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