BioSystems 65 (2002) 123 138 www.elsevier.

com/locate/biosystems

From molecular biology to nanotechnology and nanomedicine

Katarzyna Bogunia-Kubik a,b,*, Masanori Sugisaka a
b

Faculty of Engineering, Oita Uni6ersity, Dannoharu 700, 870 -1192 Oita, Japan L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53 -114 Wroclaw, Poland
a

Received 30 April 2001; received in revised form 4 February 2002; accepted 10 February 2002

Abstract Great progress in the development of molecular biology techniques has been seen since the discovery of the structure of deoxyribonucleic acid (DNA) and the implementation of a polymerase chain reaction (PCR) method. This started a new era of research on the structure of nucleic acids molecules, the development of new analytical tools, and DNA-based analyses. The latter included not only diagnostic procedures but also, for example, DNA-based computational approaches. On the other hand, people have started to be more interested in mimicking real life, and modeling the structures and organisms that already exist in nature for the further evaluation and insight into their behavior and evolution. These factors, among others, have led to the description of articial organelles or cells, and the construction of nanoscale devices. These nanomachines and nanoobjects might soon nd a practical implementation, especially in the eld of medical research and diagnostics. The paper presents some examples, illustrating the progress in multidisciplinary research in the nanoscale area. It is focused especially on immunogenetics-related aspects and the wide usage of DNA molecules in various elds of science. In addition, some proposals for nanoparticles and nanoscale tools and their applications in medicine are reviewed and discussed. 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: DNA analyses; DNA computation; Articial cells modeling; Nanoparticles; Nanomedicine

1. Nanotechnology operations on DNA molecules The discovery of polymerase chain reaction (PCR) (Mullis et al., 1986; Saiki et al., 1986) opened a new area of biological research. The
* Corresponding author. Tel./fax: + 81-975-54-7841. E -mail address: bogunia-kubik@bigfoot.com (K. BoguniaKubik).

impact can be observed not only by the great progress in the eld of molecular biology, but also in many achievements in other related elds of science. Molecular biology techniques have been implemented successfully in biology, biotechnology, medical science, diagnostics, and many more. The introduction of PCR resulted in improving the old, and designing the new laboratory devices for PCR amplication and analysis of amplied deoxyribonucleic acid (DNA) fragments. In paral-

0303-2647/02/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 2 6 4 7 ( 0 2 ) 0 0 0 1 0 - 2

124

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

lel to these efforts, the nature of DNA molecules, their construction, and their potential use as a computation medium have attracted many researchers. In addition, some studies concerning mimicking living systems, as well as developing and constructing articial nanodevices, such as biomolecular sensors and articial cells, have been conducted. These factors, among others, are the origins of a new nanoscale technology domain.

1.1. Technical tools for micro - and nanoscale DNA analysis

Most research, especially in biological sciences, cannot be conducted without specialized laboratory tools and equipment. Progress in the scientic domain is accompanied, if ever possible, by the proposals in the technical domain. Such coincidence can be observed between recent achievements in molecular biology and in the development of equipment to perform analyses and diagnostic procedures based on DNA (or ribonucleic acid (RNA)). Particular attention should be paid to PCR and the electrophoresis apparatus, which have been improved signicantly and introduced into routine scientic laboratory work. Electrophoresis apparatuses are used for identication of the products of PCR amplication. They are built on the principle that in a microenvironment with 5 B pH B 9, DNA molecules are charged negatively and they are able to migrate in an electric eld. When analysis is performed in a gel, the dynamics of the migration depend mostly on the DNA molecules size (Sambrook et al., 1989). Originally, PCR amplications were performed in water thermocyclers in a reaction volume of 100 ml employing 30 PCR cycles for over 3 h. But in routine laboratory work, water thermocyclers have been replaced by more modern machines that use Peltier elements and heating blocks. These machines allow PCR amplications to be performed in 200 ml tubes in a nal reaction volume of 10 ml using 30 PCR cycles for about 1.5 h (i.e. PTC-200 DNA Engine Line Gradient Feature, MJ Research Inc., Waltham, MA, USA). Currently, even more precise devices are available.

One of the rst microdevices to both amplify and detect PCR products without intermediate steps was reported in Wooley et al. (1996). This device, composed of a silicon reaction chamber attached to a glass capillary electrophoresis separation channel, had a 10 ml volume and performed amplications in 15 min. Since then, a great number of different microuidic PCR devices have been fabricated in silicon (Chuadhari et al., 1998; Daniel et al., 1998; Northrup et al., 1998; Wilding et al., 1998), glass (Taylor et al., 1998; Waters et al., 1998), silicon glass hybrids (Cheng et al., 1996), and in fused silica capillaries (Zhang et al., 1998). During the BioMENS and Biomedical Nanotechnology World 2000 conference (reviewed by Wooley (2001)) many interesting machines for biochemical analysis were presented. These included mainly micromachines allowing different DNA analyses to be performed, e.g. PCR amplication, electrophoresis, and sequencing. One of them was a high-throughput biochemical microprocessor is able to perform rapid and parallel DNA sequencing on 96 samples to 500 bases, corresponding to a throughput of 100 kb/h. An integrated device for PCR amplication and electophoretic analysis was also interesting. This was a fully integrated system in glass for the manipulation, amplication, and capillary electrophoresis separation of submicroliter volumes of DNA (Lagally et al., 2000). In this system, the use of a low-volume reactor with 280 nl PCR chambers and thin-lm heaters permitted thermal cycle times as fast as 30 s. The amplied product was labeled with an intercalating uorescent dye and injected directly into a microfabricated capillary electrophoresis system. The reaction could start with as few as 20 DNA template copies per microliter (5 to 6 per chamber). During the same conference, miniaturized highspeed thermal cycling chambers were shown. These chambers were able to complete 10 PCR cycles in 30 s and successfully amplify a DNA sample in fewer than 4 min. 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

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

125

described (Wooley et al., 2000). This technique was based on the hybridization of labeled 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 (Wooley et al., 2000). In addition, a novel method for electrochemical detection of DNA hybridization with the use of conductive hydrogels was proposed. These micro- and nanotechnological tools are being used to sequence genomes and diagnose diseases. The analyzers mentioned above may allow much faster, more specic, 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). Employing more efcient machines may also improve and shorten the time of analyses, which can be of particular importance, e.g. when searching for suitable donors for transplantation for patients suffering from hematological disorders (Bogunia-Kubik and Lange, 2000), where the time factor is critical.

1.2. Computation at DNA le6el

In recent years a great interest in biologically inspired systems has been seen among researchers developing new computer techniques. One branch is based on the fact that genetic information is stored in nucleic acid molecules. Moreover, these DNA molecules possess massively parallel processing capabilities and are easy to manipulate in a genetic engineering laboratory. These facts inspired hopes that DNA-based computers, if built, might handle millions of operations in parallel and solve hard computational problems in a reasonable amount of time (Kubik and BoguniaKubik, 2002).

1.2.1. In 6itro DNA computation The principle of computing with an ordinary computer relies on transmitting logical signals through an especially designed network of paths, gates, and connections. These signals have the

form of electrical impulses, whose voltage level is interpreted as a logical 0 or 1. Computer programs are collections of words consisting of such binary values that, as electrical impulses, are transmitted through the computers electronic control devices. DNA-based computation has a quite different nature. In the case of DNA, words are composed of specic sequences of DNA oating in a medium (there are no conductor paths), and the process of computing has the form of chemical reactions. With a DNA computer, computation requires synthesizing particular sequences of DNA in a separate process (according to the problem) and letting them react in a test tube (reactions are performed extracellularly in solution or afxed to a surface of glass or silicon). These kinds of DNA-based computational approaches have been called in vitro DNA computation. The practical possibility of using in vitro DNA computation was demonstrated by Adelman in his pioneering work (Adelman, 1994). He used DNAbased computation to solve a seven-node Hamiltonian path problem (HPP). The HPP is dened as follows: given a graph, is there a path through the graph, which visits each vertex precisely once? HPP belongs to a class of NP-complete problems. The 7-node case of HPP is trivial to solve, so the experiment performed by Adelman must be regarded as an illustration of the potential of DNA computing. At rst, Adelman constructed two sets of 20-nucleotide long sequences representing particular vertices and particular connections between two vertices in a 7-node graph, respectively. In each vertex representation, ten rst and ten last nucleotides were unique and were interpreted as sequences representing the input and output of the vertex, respectively. Sequences representing connections were constructed from the sequences that were complementary to the outputs and inputs of the adequate vertices. Adelman then started the calculation part of the experiment. He used PCR to amplify all possible connections/paths, which began with vertex 0 and ended with vertex 6. Next, the electrophoresis of amplication products led him to the separation of sequences with length corresponding to the

126

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

path that passed through all vertices. Further steps of the analysis allowed the selection of those sequences, which represented paths without repeated vertices. This part of the experiment needed serial hybridizations with the use of adequate panels of nucleotide sequences immobilized on magnetic beads. Any sequences that remained after that analysis constituted solutions of the problem. Thus to solve the HPP, Adelman employed a synthesis of DNA fragments, amplication with the use of PCR, electrophoresis, probing, and nally sequencing the remaining DNA fragments selected throughout this multistep analysis. Following in the path of Adelmans experiment, Lipton (1995) proposed a series of experiments to tackle NP-complete satisfaction problems (SAT). The SAT problem is dened as follows: given a Boolean formula F, is there an assignment of values to the variables of F so that F is true? Recently, Liu et al. (2000) published their work on DNA computing on surfaces. They proposed a method of solving the NP-complete SAT problem by attaching candidate DNA oligonucleotides to a support, and screening using successive hybridization operations. The success of experiments using DNA fragments to solve calculation problems has generated much more interest in the scientic world about the potential properties and usage of DNA molecules. Seeman and co-workers (Seeman, 1999; Seeman et al., 1998) performed studies on DNA molecules analyzing, among other things, their secondary and tertiary structure and creating specic topologies, shapes, and arrangements of ligated DNA strands. They also proposed the utilization of DNA for a practical solution of cumulative XOR calculations. For this purpose, they used appropriately constructed DNA fragments and employed a number of molecular biology techniques, including ligation, PCR, digestion of PCR products with restriction enzymes, and electrophoresis. The next examples of a practical DNA implementation were demonstrated by Ouyang et al. (1997) and van Noort and McCaskill (2001). In both cases, the objective was to solve the NP-

complete maximum clique problem (MCP). The MPC is dened as follows: given an undirected graph, nd the largest set (maximum clique) of its mutually adjacent vertices. The method of Ouyang and co-workers (Ouyang et al., 1997) was based on construction of 64 DNA doublestranded molecules that were analyzed by selective digestion with restriction enzymes. To solve the MCP, van Noort and McCaskill (2001) constructed not only a set of appropriate DNA sequences, but also an entire microow reactor, where particular hybridization reactions of analyzed DNA strands with selecting DNA sequences immobilized on the paramagnetic beads occurred. Each analyzed sequence offered a binary encoding of particular subgroups of nodes in the graph considered, where 0 (or 1) was represented by the whole fragment of a DNA sequence (not only by a single nucleotide base pair). The end effect of this selection (the solution) was observed by CCD cameras and a microscope. Some other proposals of DNA computation have been based on the usage of specically designed circular DNA molecules (DNA plasmids) as a computational tool. These methods were used, for example, for analysis of the NP-complete algorithmic problem of computing the cardinal number of a maximal independent subset (MIS) of a graph (Head et al., 2000) and solving the graph coloring problem (GCP) (Kubik et al., 2001). In both studies, plasmids contained specially inserted DNA sequences so that each one was bordered by a characteristic pair of restriction enzyme sites. The calculation steps involved employing of a number of restriction endonuclease treatments to the computational plasmid. In the case of the MIS problem, the calculations ended with a tube containing plasmids representing all independent subsets of the graph (subsets of vertices not connected by the edge). A solution to GCP (assignments of colors to graph vertices such that no two vertices connected by an edge are painted with the same color while the total number of colors is minimized) was found by extending this procedure with some additional restriction enzyme treatments and serial hybridization steps (Kubik et al., 2001).

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

127

1.2.2. In 6i6o DNA computation DNA computation as described by the pioneer work of Adelman and many followers is currently called in vitro DNA computation. But researchers have also tried to nd another method for DNAbased computation. The idea is to use the properties of living cells and organisms, more exactly, to use DNA molecules and solve computational problems within a living cell. In this way, in vivo approaches to DNA computation have been introduced. In one study, Matsuno et al. (2001) proposed an in vivo method for computation of two logic functions: T (X ) = X, and F (X ) = X. They demonstrated the method using the biological properties of Escherichia coli bacteria, and their ability to synthesize b-galactosidase (controlled by lac-operon) in the presence of an induction factor (IPTG). So, two biological processes, gene expression and protein synthesis, were involved in the computation process (Matsuno et al., 2001). There are also other reports pointing out the possibility of in vivo computation. For example, Gardner et al. (2000) presented another computational approach in E. coli that proposed the construction of a genetic toggle switch forming a cellular memory unit. Eng (1999) proposed solving the 3CNF-SAT problem (a special case of SAT problem) with an in vivo algorithm. Other interesting in vivo DNA computation proposals on constructing DNA or RNA/rybozyme based molecular switches come from the study of Gardner et al. (2000) and Soukup and Breaker (1999). 1.2.3. Contrary 6iews on molecular computation The implementation of in vitro and in vivo molecular computation (recently based also on RNA (Cukras et al., 1999; Faulhammer et al., 2000)) illustrates how knowledge coming from molecular biology and genetics can be implemented to solve hard computational problems. Although the practical benets of DNA-based computational schemes are still questionable and the vast majority of work to date has been theoretical, there have been many allusions to potential uses of this emerging computational paradigm. It has been suggested that DNA/ biomolecular computation is faster, uses less en-

ergy, has a greater potential for information storage, and the ability to handle much larger numbers of operations in parallel than conventional computing (Baum, 1995; Chen and Wood, 2000). According to Baum (1995), the memory of molecular computers may be of greater capacity than that of a human brain. Although the technical potential of DNA-based computation looks very promising, the practical usage, including the high costs operations performed, seems to be limited. DNA-based calculations require a large number of specically encoded nucleotides (Bunow, 1995). It has been calculated that a HPP with 23 nodes would need kilogram quantities of DNA (Lo et al., 1995), but the amount of DNA required to solve a 70-node problem would equal over 1000 kg of nucleic acids (Linial and Linial, 1995). In addition, DNA computation might be erroneous and might cause technical problems as has been investigated preliminarily (Aoi et al., 1999) by analyzing the errors that may occur during the ligation process of DNA sequences, and discussed by Cox et al. (1999). Moreover, to date, no large computationally complex problem has been encoded in DNA and solved by molecular biology (Rozen et al., 1996). Researchers in this eld look for problems that could be solved by molecular computers rather than at the method of analysis. DNA computation is based generally on the generation of all possible DNA-encoded candidate answers followed by a good candidate search and nally the interpretation of the results, for example, by candidate sequencing. This technique has not been used strictly to solve arithmetic problems, but rather to help to nd a solution or a set of solutions that meet specic criteria from the pool of possible combinations. However, there has been an attempt to use DNA to sum binary integer numbers (Guarnieri et al., 1996; Wasiewicz et al., 2000). Thus, performing calculations employing biomolecules and using genetic engineering technology, although now questionable, may soon become the most widely used tool for computation.

128

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

1.3. Modeling of life articial cells

We have indicated that biological tools (DNA molecules, enzymes, and different kind of biological reactions), with some limitation, can be used to solve computational problems. In this way, biology helps mathematical sciences. However, even when employing very modern technical tools and high-resolution devices, it is not possible to observe in great details the processes ongoing within living cells or look deeply at and analyze the mechanisms of cell cell interactions. For that reason, mathematical tools and computer software can be employed, for the description and modeling of structures and organisms already existing in nature. This theoretical research may help in the evaluation and give some insight into the behavior and evolution of biological systems. A few examples of mathematical models, including the simulation of gene expression, protein production, and interactions of the cells, will be presented below.

1.3.1. Models of li6ing organisms On the basis of a bacteria genome, Tomita et al. (1999) constructed a model of a hypothetical cell with only 127 genes being sufcient for transcription, translation, energy production, and phospholipid synthesis. Most of the genes were taken from Mycoplasma genitalium, the organism having the smallest-known chromosome, whose complete 580 kb genome sequence was determined in 1995. They developed the E-cell system, which is a computer software environment for modeling and simulation of the cell, in order to understand how all the cellular proteins work collectively as a living cell system. The goal of this study is, by attempting to understand the dynamics in living cells, to predict consequences of changes introduced into the cell and/or its environment, e.g. knocking out genes or altering available metabolites. It is also worth noting work on a more complex organism, the fruit y Drosophila, for simulation of Drosophila embryogenesis (Hamahashi and Kitano, 1998) or leg formation (Kyoda and Kitano, 1999), as examples of simulations of gene interactions and expression.

1.3.2. Mathematical modeling of gene networks DNA duplication (DNA synthesis), transcription (synthesis of mRNA on the DNA template), and translation (protein production) are processes that take place in any living cell. A number of models have been proposed in order to analyze and simulate interactions between genes (gene networks) and the control of gene transcription and expression processes gaining insights into the static and dynamic behavior of complex biological systems. These, for example, have been shown and reviewed by Smolen and co-workers (Smolen et al., 2000, 2000a). The regulatory effect of one gene product on the expression of other genes were analyzed by Vohradsky (2001). Moreover, algorithms for inference of genetic networks (called AIGNET systems), introduced by Maki et al. (2001), were implemented for analysis of the interference of a genetic network composed from just 30 genes. This model allows the analysis of the different patterns of gene expression, including some kinds of gene perturbations, such as disruption or over expression. In addition, these kinds of analyses may lead to the identication of DNA sequences that may serve as potential targets of antisense oligonucleotides and sequences of genes in, discussed later, antisense and gene therapies (Zanders, 2000). 1.3.3. Mathematical model of an immune system Cell interactions and co-operations constitute the bases of many mathematical simulations. Studies have been undertaken to describe and analyze the relations between T and B cells of the immune system that will be described here in more detail. B and T cells are two major classes of lymphocytes playing an important role in the immune system response. B cells, B lymphocytes, upon activation by an antigen, differentiate into cells producing antibodies. T cells are subsets of lymphocytes dened by their development in the thymus and by their heterodimeric receptors (T cell receptor (TCR)) that can recognize the antigen presented by MHC (major histocompatibility complex) molecules. MHC molecules are expressed on antigen presenting cells (such as macrophage, B lymphocytes or dendritic cells).

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

129

Two classes of MHC molecules present antigenic peptides to T lymphocytes. MHC class I molecules present peptides generated in cytosol to CD8 T cells, and MHC class II molecules present peptides degradated in cellular vesicles to CD4 T cells. CD4 T cells, T helper cells, can help B cells make antibodies in response to an antigenic challenge. The other kind of T lymphocytes, cytotoxic T cells, which are mostly CD8 T cells, can kill other cells. This is important in host defense against cytosolic pathogens. Th1 and Th2 cells, and Tc1 and Tc2 cells, are the next sets of Th and Tc cells, respectively. The Th1 (inammatory CD4 cells) subtype associates with cell-mediated immunity, while the Th2 (helper CD4 cells) type promotes B cell growth and antibody production. Agrawal and Linderman (1996) employed mathematical modeling to investigate the effect of antigen processing and presentation on the Th cell response. Using two mathematical models, they generated theoretical curves in order: (i) to relate the external antigen concentration to the number of MHC peptide complexes present on APCs, and (ii) to relate the number of MHC peptide complexes on the APC to the number of bound TCRs on the Th cell. Our model showed that modications to peptide antigen resulted in altered MHC peptide and/or TCR/MHC peptide afnities and the associated kinetic bindings. The second model predicted that: (i) the TCR/MHC peptide afnity alone has a dramatic effect on the number of bound TCRs at smaller but not larger afnity; (ii) the number of MHC peptide complexes required to obtain a particular number of bound TCRs varies over a range, depending on the TCR MHC afnity; and (iii) that a large value of one afnity (e.g. a large MHC peptide afnity) can compensate a small value of another one (e.g. a small TCR/MHC peptide afnity). In the same year, Carneiro et al. (1996) described a model of the immune network with B T cell co-operation, where B cell activation was dependent on T cell help, and activated T cells were downregulated by engagement of their TCRs by anti-TCR soluble immunoglobulines. They tested their model by examining several prototypic situations with a small number of T and B cell clones. (A clone is a population of cells

derived from a common progenitor.) One of the latest reports from this group describes another proposal of computer simulation used for analysis of interactions between regulatory and target T cells, using the mechanism of T cell mediated suppression (Leon et al., 2001). Recently, Tarakanov and Dasgupta (2000) proposed a model of an articial immune system, referred to as a formal immune system (FIS). This mathematical approach is based on the features of antigen antibody bindings. It provides a mathematical description of B and T cells, with a simulation of B and T cell interactions and protein bindings. Coetano et al. (1998) dened a more precise computational model for the dynamics of T helper lymphocytes subpopulations (Th1 and Th2 cells) to study multiple responses and cross-regulatory points, and T cell antigen presenting cell interaction. In this study, the authors focused especially on pathways leading to T cell differentiation into Th1 and Th2 cells. These mechanisms play a major role in the immune system oncogeny. T helper cells and the inuence of cytokines on their regulation were the aim of another computational model proposed by Yates et al. (2000). Mathematical modeling of the immune system and cell interaction allows investigation of the immunological response in health and disease. For example, some studies have been carried out to analyze the mechanism of tumor cells interaction (Bach et al., 2001), tumor angiogenesis (Levine et al., 2001), and development (Kozusko et al., 2001; Swanson et al., 2001).

2. Nanotechnology materials, articial structures and devices The word nanotechnology is used to describe many types of research where the characteristic dimensions are smaller than about 1000 nm. This denition covers a wide range of different scientic elds, from lithography (with line widths less than one micron) to nanomachines and nanorobots. The term nanotechnology can be understood as altering individual atoms and molecules at a pre-

130

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

cise location, either chemically or physically. Nanotechnology also seeks to develop devices that can scan and to manipulate objects at near atomic scale, such as the atomic force microscope (Liaw et al., 1998), the scanning tunnel microscope (Permiakov et al., 1998), the laser force microscope, laser tweezers (Kellermayer et al., 2000), or liquid chromatography mass spectrometry devices (Guetens et al., 2000). Nanotechnology (in other words molecular nanotechnology, molecular manufacturing) also comprises the building of nanostructures and manufacturing nanometer-scale objects. Therefore, DNA computation (as manipulation of nanoscale particles), different kinds of microscopy, manipulation of molecules, constructing nanometre resolution diagnostic and analytical devices, and nanomachines and nanorobots, can all be described by this term nanotechnology. To date, several nanoscale devices have been described in the literature. Among them there are a few composed of nucleic acids, e.g. a nanomechanical DNA device (Mao et al., 1999) and nucleic acid molecular switches based on rybozyme structure (Soukup and Breaker, 1999). Some interesting proposals have been derived from Drexler (1981, 1986, 1992) and his followers (such as Merkle (1996) and Freitas, see the references below) working in the eld of molecular nanotechnology and nanorobot construction. They have described nanomachines that could nd practical implementation in medicine. One of them is a model of an articial red blood cell (Freitas, 1996b, 1998).

as an articial erythrocyte, duplicating the oxygen and carbon dioxide transport functions of red cells, mimicking the action of natural hemoglobin-lled red blood cells. It is expected to be capable of delivering 236 times more oxygen per unit volume than a natural red cell. Specially installed equipment enables this device to display many complex responses and behaviors. Additionally, it has been designed to draw power from abundant natural serum glucose supplies, and thus is capable of operating intelligently and virtually indenitely, whilst red blood cells have a natural lifespan of 4 months.

2.2. Articial cell membranes

This is the concrete implementation of an articial cell organelle coming from Cornell and coworkers (Cornell et al., 1997). They developed a synthetic biosensor that imitates nerve cell membranes. This articial membrane, an ion-based nanomachine, is composed of the following elements: membrane-forming molecules that are tethered chemically to the gold surface; simple ion channels within the membrane that facilitate the transport of ions (like Na+, K+); a reservoir space between the surface and the membrane to store ions; and receptors such as antibodies attached to the membrane to recognize target molecules. The detection mechanism operates by binding the target molecule, which alters the population of conduction ion channel pairs within the tethered membrane. This results in a change in the membrane conduction. The analysis is based on the measurement of changes in the membrane conduction. This is already a commercial product supplied by the Ambri Ltd. (Chatswood, NSW, Australia) that can be used, for example, in pharmaceutical research or for rapid medical testing.

2.1. Mechanical articial red cells

The articial mechanical red blood cell, called respirocyte, was designed by Freitas (1996a, 1998). This was the rst detailed design study of a specic medical nanodevice (of the kind proposed by Dexler in Nanosystems). The proposed respirocyte measures about 1 mm in diameter and just ows along the bloodstream. It is a spherical nanorobot made of 18 billion atoms. The respirocyte is equipped with a variety of chemical, thermal, and pressure sensors and an onboard nanocomputer. This device is intended to function

2.3. Articial nanostructures that can interact with and replace natural biological materials
In the report from one of the meetings of American scientists (American Chemical Society ProSpectives, Berkeley, CA, USA, 2001) Taton (2001) presents a very intriguing proposal of an articial bone relying on designing the synthetic

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

131

substitutes of collagen. Research on designing self-assembling, synthetic substitutes for collagen has been conducted by a group of Stupp at Northwestern University, Evanston, IL. They proposed an articial material, composed of amphiphilic molecules bearing a long hydrophobic alkyl group on one end and ahydrophilic peptide on the other that was able to spontaneously assemble into cylindrical structures that resemble collagen brils. Moreover, these cylinders guided the formation of hydroxyapatite crystallites. What is even more important, they formed crystallites characterized with orientations and sizes similar to those in natural bone. Taton emphasized that these observations lead to a general question: would synthesized nanomaterials be able not only to replicate the properties of their natural equivalents (cell membranes, tissues and bone marrow), but also prompt biological systems to build up on these materials, and to produce self-assembling structures? Studies on such a nanostructures lead to promising materials with potential uses as implants and therapies. Moreover, they may someday show how the cells interact with nanometer-sized objects in their own world.

3. Nanomedicine another implementation of nanotechnology From nanotechnology it is only one step to nanomedicine, which may be dened as the monitoring, repair, construction, and control of human biological systems at the molecular level, using engineered nanodevices and nanostructures. It can also be regarded as another implementation of nanotechnology in the eld of medical science and diagnostics. One of the most important issues is the proper distribution of drugs and other therapeutic agents within the patients body.

3.1. Nanoparticles as carriers of therapeutic molecules

Targeting the delivery of drugs to diseased lesions is one of the important aspects of the drug delivery systems. To convey a sufcient dose of drug to the lesion, suitable carriers of drugs are

needed. Nano- and microparticle carriers have important potential applications for the administration of therapeutic molecules. Liposomes have been used as potential drug carriers instead of conventional dosage forms because of their unique advantages, which include the ability to protect drugs from degradation, target the drug to the site of action, and reduce the toxicity of side effects (Knight, 1981). However, developmental work on liposomes has been limited, due to inherent problems such as low encapsulation efciency, rapid leakage of water-soluble drugs in the presence of blood components, and poor storage stability. In some cases, nanoparticles are more efcient drug carriers than liposomes due to their better stability (Fattal et al., 1991) and possess more useful control release properties. These are the reasons why many drugs have been associated with nanoparticles (e.g. antibiotics, antiviral and antiparasitic drugs, cytostatics, vitamins, protein and peptides, including enzymes and hormones (please note the references for this paragraph of the paper)). Nanoparticles are dened as being submicronic ( B 1 mm) colloidal systems generally made of polymers (biodegradable or not). They were rst developed in the mid 1970s by Birrenbach and Speiser (1976). Nanoparticles generally vary in size from 10 to 1000 nm. The drug is dissolved, entrapped, encapsulated, or attached to a nanoparticle matrix. Depending upon the process used for the preparation of nanoparticles (reviewed by Kumar, 2000; Lambert et al., 2001; Soppimath et al., 2001), nanospheres or nanocapsules can be obtained. Nanocapsules are vesicular systems in which the drug is conned to a cavity (an oil or aqueous core) surrounded by a unique polymeric membrane. Nanospheres are matrix systems in which the drug is physically and uniformly dispersed throughout the particles. In recent years, biodegradable polymeric nanoparticles have attracted considerable attention as potential drug delivery devices, this is in view of their applications in controlling drug release, their ability to target particular organs/tissue, as carriers of oligonucleotides in antisense therapy, DNA in gene therapy, and in their ability to deliver proteins, peptides and genes through

132

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

oral administration (Langer, 2000). These applications of nanoparticulate systems will be discussed below in more detail.

3.1.1. Antisense therapy Nucleic acids can be used not only to diagnose and monitor, but also to prevent and cure diseases, as they constitute the bases of antisense and gene therapies. Antisense oligonucleotides have emerged as potential gene-specic therapeutic agents and are currently undergoing evaluation in clinical trials a variety of diseases. These include advanced carcinoma (Cunningham et al., 2001; Rudin et al., 2001) and non-Hodgkins lymphoma (Waters et al., 2000; Gewirtz, 1999). Theoretically, an antisense oligonucleotide is a short fragment (15 20 bp) of deoxynucleotides characterized with a sequence complementary to a portion of the targeted mRNA. The aim of the antisense strategy is to interface with gene expression by preventing the translation of proteins from mRNA. There are a few mechanisms of mRNA inactivation (Lambert et al. 2001), including (i) sterical blocking of mRNA by antisense binding and destruction antisense-mRNA hybrids by RnaseH enzyme, (ii) formation of triple helix between genomic double-stranded DNA and oligonucleotides, or (iii) the cleavage of target RNA by ribozymes. Antisense oligonucleotides are molecules that are able to inhibit gene expression, being therefore, potentially active for the treatment of viral infections or cancer. However, the problems such as the poor stability of antisense oligonucleotides versus nuclease activity in vitro and in vivo, and their low intracellular penetration have limited their use in therapeutics (Loke et al., 1989; Yakubov et al., 1989). In order to increase their stability, to improve cell penetration and also avoid non-specic aptameric effects (leading to non-specic binding of antisense oligonucleotides), the use of particulate carriers such as liposomes or nanoparticles, has been considered. It has also shown interesting potentialities to bind and deliver oligonucleotides in an efcient manner (Fattal et al., 1998). Very recently, it was reported that antisense oligonucleotides can be encapsulated in nanocapsules with a size of 350 9 100 nm.

A formulation of these capsules might have special importance for oligonucleotide delivery. The rst experiments on the treatment of RAS cells expressing the point-mutated Ha-ras gene were promising (Lambert et al., 2001). In addition to the nanoparticle preparation, new techniques that enable analysis of their proper delivery are being developed. For example, to address the crucial problem of oligonucleotide degradation, an original assay method was proposed allowing, in the polyacryamide gels, quantication of the amount of undegradated oligonucleotides (16 mer oligothymidilate) in tissues such as liver and plasma (Aynie et al., 1996).

3.1.2. Gene therapy and administration of DNA 6accines Gene therapy is a recently introduced method for treatment or prevention of genetic disorders based on delivery of repaired, or the replacement of incorrect, genes. It is aimed at treating or eliminating the causes of disease, whereas most current drugs treat the symptoms. There is the wide range of target cells and diseases, like cancer, infectious, cardiovascular, monogenic (e.g. hemophilias) diseases, and rheumatoid arthritis, for which clinical studies are ongoing (Mountain, 2000). In fact, the rst disease approved for gene therapy treatment was adenosine deaminase (ADA) deciency, and the rst patient was treated in 1990. Recently, Onodera et al. (1998) reported the use of ADA copy DNA (cDNA) for the treatment of severe immunodeciency (SCID) patients. In addition, one of the latest reports on gene therapy demonstrates the successful treatment of patients with hemophilia B, with a defect in a gene encoding blood coagulation factor IX (Kay et al., 2000) and patients with hemophilia A having a defect in a gene that encodes factor VIII (Roth et al., 2001). In these cases, patients broblasts transfected with a plasmid containing sequences of the factor VIII gene (hemophilia A treatment) and adeno-associated viral vectors expressing human factor IX (hemophilia B) were used for gene transfer. Application of nanotechnological tools in human gene therapy has been reviewed widely by Davis (1997). He described non-viral vectors based on nanoparticles (usually

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

133

50 500 nm in size) that were already tested to transport plasmid DNA. He emphasized that nanotechnology in gene therapy would be applied to replace the currently used viral vectors by potentially less immunogenic nanosize gene carriers. So delivery of repaired genes, or the replacement of incorrect genes, are elds where nanoscaled objects could be introduced successfully. On the other hand, genetic immunization with DNA vaccines has emerged as one of the most promising applications of non-viral gene therapy (Ulmer et al., 1996; Dubensky et al., 2000), having a number of the potential advantages over conventional vaccines. These include: (i) the high stability of plasmid DNA, (ii) low manufacturing costs, (iii) lack of infection risk associated attenuated viral vaccines, (iv) the capacity of target multiple antigens to one plasmid, and (v) the ability to elicit both humoral and cellular immune responses. Until recently, intramuscular injection was the primary route of administration of DNA vaccines. As an alternative to intramuscular administration of plasmid DNA, researchers have been investigating targeting plasmid DNA to the skin using intradermal needle injection, needlefree jet injection devices, the gene gun, or recently topical delivery (Cui and Mumper, 2001) of formulated plasmid in the form of a patch, cream, or gel. The latter method may provide many advantages in terms of cost and patient compliance (Shi et al., 1999). Among other nanoparticles, chitosan, a biodegradable polysaccharide comprise of primarily D-glucosamine repeating units, has been proposed by several groups as an alternative non-viral delivery system for plasmid DNA. Selective chitosan polymers and chitosan oligomers have been found to efciently condense plasmid DNA and to transfect several different cell types in vitro and in the intestines, colon, nose, and lung (Erbacher et al., 1998; Cui and Mumper, 2001).

peptides and proteins by the oral route have attracted considerable attention as being much easier and more acceptable. Unfortunately, this route cannot be used with most proteins and peptides, due to both the degradation of these molecules within the intestine and their poor uptake across the intestinal wall. To potentially overcome these problems, it has been shown that it is possible to utilize the uptake mechanism of vitamin B12 to enhance the oral uptake of various peptide and protein pharmaceuticals. In particular, molecules such as luteinizing hormone releasing hormone (LHRH) analogues, a-interferon, erythropoietin, and granulocyte colony stimulating factor (G-CSF) have been studied. These pharmaceuticals have been linked covalently to the vitamin B12 molecule (Russell-Jones, 1998; Russell-Jones and Alpers, 1999). The system relies upon the natural uptake mechanism for vitamin B12 to co-transport peptides and proteins linked to vitamin from the intestine into the circulation. To maximize the potential of the delivery system, the pharmaceutical is being incorporated within biodegradable nanoparticles, and coated with vitamin B12 (Russell-Jones et al., 1999). This has advantages of protecting the pharmaceutical from proteolysis within the intestine, of amplifying the uptake capacity of the oral delivery system, and of eliminating the need for conjugation of pharmaceuticals to vitamin B12. There have also been other proposals utilizing the pre-existing mechanisms of molecule delivery within the body, such as drug targeting biodegradable nanoparticles coupled to folic acid (Stella et al., 2000).

3.1.3. Treatment of patients with peptide and protein pharmaceuticals Currently, this treatment is performed mostly by injection, with accompanying patient discomfort, increased medical costs, and reduced patient compliance. Therefore, the systems of delivery of

3.1.4. Other applications In addition to oral administration (Langer, 2000), the use of nanoparticles for nasal (Illum et al., 2001) and ophthalmic delivery of drugs (Bourlais et al., 1998; de Campos et al., 2001) has been investigated. Nanoparticles have enabled crossing the blood brain barrier that represents an insurmountable obstacle for a large number of drugs, including antibiotics, antineoplastic agents, and a variety of central nervous system active drugs, especially neuropeptides (Kreuter, 2001; Schroeder et al., 1998). Furthermore, nanosize carriers of such molecules as vitamins, vitamin A

134

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138

(Jenning et al., 2000) and E (Dingler et al., 1999), have potential applications in dermatology and cosmetics.

4. Towards the future Medical diagnosis with proper and efcient delivery of pharmaceuticals are the medical elds where nanosize materials have found practical implementations. However, there are several other intriguing proposals for practical applications of nanomechanical tools into the elds of medical research and clinical practice. Such nanotools still await construction, and at present they are rather more like a fantasy. Nevertheless, they might be very helpful, and become a reality in the near future. One function of nanodevices in medical sciences could be the replacement of defective or incorrectly functioning cells, such as the respirocyte proposed by Freitas (1996a, 1998). This articial red blood cell is theoretically able to provide oxygen and can do it even more effectively than an erythrocyte. It could replace defective natural red cells in blood circulation. An onboard nanocomputer and numerous chemical and pressure sensors enable complex device behaviors that are remotely reprogrammable by the physician via externally applied acoustic signals. Primary applications of respirocytes may include transfusable blood substitution, partial treatment for anemia, prenatal/neonatal problems, and lung disorders. This articial respirocyte could support oxygen distribution, improving the levels of available oxygen despite reduced blood ow. Thus the next application phase of nanomachines could be providing metabolic support in the event of impaired circulation. It has also been postulated that nanomachines could distribute drugs within the patients body. Such nanoconstructions could deliver medicines to particular sites, making more adequate and precise treatment possible (Fahy, 1993a,b; Triggle, 1999). Such devices would have a small computer, several binding sites to determine the concentration of specic molecules, and a supply of some poison that could be released selectively. Similar

machines equipped with specic weapons could be used to remove obstructions in the circulatory system or identify and kill cancer cells. The other important application of nanotechnology relates to medical research and diagnostics. Nanorobots, operating in the human body, could monitor levels of different compounds and store that information in internal memory. They could be used to rapidly examine a given tissue location, surveying its biochemistry, biomechanics, and histometric characteristics in greater detail. This would help in better disease diagnosing (Freitas, 1996a; Lampton, 1995). The use of nanodevices would give the additional benets of reduced intrusiveness, increased patient comfort, and greater delity of results, since the target tissue can be examined in its active state in the actual host environment. The use of nanoscale machines cruising through our bodies, attacking viruses and diseases, killing cancer cells, and repairing damaged cells and tissues, may seem now to be science ction. However, in researchers imaginations such machines have already appeared and now there are only a few steps to make these designs come true. Acknowledgements The authors are grateful to Dr J. Johnson and Dr T. Kubik for their help in the nal preparations of the manuscript. References
Adelman, L.M., 1994. Molecular computation of solutions to combinatorial problems. Science 266, 1021 1024. Agrawal, N.G.B., Linderman, J.J., 1996. Mathematical modeling of helper T lymphocyte/antigen-presenting cell interactions: analysis of methods for modifying antigen processing and presentation. J. Theor. Biol. 182, 487 504. Aoi, Y., Yoshinobu, T., Tanizawa, K., Kinoshita, K., Iwasaki, H., 1999. Ligation errors in DNA computing. BioSystems 52, 181 187. Aynie, I., Vauthier, C., Foulquier, M., Malvy, C., Fattal, E., Couvreur, P., 1996. Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity counter for the evaluation of oligonucleotide-bound drug carrier. Anal. Biochem. 240, 202 209.

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138 Bach, L.A., Bentzen, S.M., Alsner, J., Christiansen, F.B., 2001. An evolutionary-game model of tumour-cell interactions: possible relevance to gene therapy. Eur. J. Cancer 37, 2116 2120. Baum, E.B., 1995. Building an associate memory vastly larger than the human brain. Science 268, 583 585. Birrenbach, G., Speiser, P., 1976. Polymerized micelles and their use as adjuvants in immunology. J. Pharm. Sci. 65, 1763 1766. Bogunia-Kubik, K., Lange, A., 2000. HLA typing in potential donors and recipients of hematopoietic stem cell transplantation. Acta Haematol. Pol. 31, 113 125. Bourlais, C.L., Acar, L., Zia, H., Sado, P.A., Needham, T., Leverge, R., 1998. Ophthalmic drug delivery systems-recent advances. Prog. Retin. Eye Res. 17, 33 58. Bunow, B., 1995. On the potential of molecular computing. Science 482 483. Carneiro, J., Coutinho, A., Faro, J., Stewart, J., 1996. A model of the immune network with B-T cell co-operation. I. Prototypical structures and dynamics. J. Theor. Biol. 182, 513 529. Chen, J., Wood, D.H., 2000. Computation with biomolecules. Proc. Natl. Acad. Sci. USA 97, 1328 1330. Cheng, J., Shoffner, M.A., Hvichia, G.E., Kricka, L.J., Wilding, P., 1996. Chip PCR: 2. Investigation of different PCR amplication systems in microfabricated silicon-glass chips. Nucleic Acids Res. 24, 380 385. Chuadhari, A.M., Woudenberg, T.M., Albin, M., Goodson, K.E., 1998. Transcient liquid cristal thermometry of microfabricated PCR vessel arrays. J. Microelectromech. Syst. 7, 345 355. Coetano, A., Grilo, A., Rosa, A., 1998. Modeling thymic selection and concominant immune responses on CD4 + T lymphocyte sub-populations. In: Adami, C., Belew, R.K., Kitano, H., Taylor, C.E. (Eds.), Articial Life VI. Proceedings of the Sixth International Conference on Articial Life. MIT Press, London, England, pp. 143 150. Cornell, B., Braach-Maksvytis, V., King, L., Osman, P., Raguse, B., Wieczorek, L., Pace, R., 1997. A biosensor that uses ion-channel switches, Nature 387, 580 584. (see also: www.ambri.com.au). Cox, J.C., Cohen, D.S., Ellington, A.D., 1999. The complexities of DNA computation. Trends Biotechnol. 17, 151 154. Cui, Z., Mumper, R.J., 2001. Chitosan-based nanoparticles for topical genetic immunization. J. Control. Release 75, 409 419. Cukras, A.R., Faulhammer, D., Lipton, R.J., Landweber, L.F., 1999. Chess games: a model for RNA based computation. BioSystems 52, 35 45. Cunningham, C.C., Holmlund, J.T., Geary, R.S., Kwoh, T.J., Dorr, A., Johnston, J.F., Monia, B., Nemunaitis, J., 2001. A Phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma. Cancer 92, 1265 1271. Daniel, J.H., Iqbal, S., Milligton, R.B., Moore, D.F., Lowe, C.R., Leslie, D.L., Lee, M.A., Pears, M.J., 1998. Silicon

135

microchamber for PCR amplication. Sens. Actuat. 71, 81 88. Davis, S.S., 1997. Biomedical applications of nanotechnology implications for drug targeting and gene therapy. Trends Biotechnol. 15, 217 224. de Campos, A.M., Sanchez, A., Alonso, M.J., 2001. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A. Int. J. Pharm. 224, 159 168. Dingler, A., Blum, R.P., Niehus, H., Muller, R.H., Gohla, S., 1999. Solid lipid nanoparticles (SLN/Lipopearls) a pharmaceutical and cosmetic carrier for the application of vitamin E in dermal products. J. Microencapsul. 16, 751 767. Drexler, K.E., 1981. Molecular engineering: an approach to the development of general capabilities for molecular manipulation. Proc. Natl. Acad. Sci. USA 78, 5275 5278. Drexler, K.E., 1986. Engines of Creation: The Coming Era of Nanotechnology. Anchor Press/Doubleday, New York. Drexler, K.E., 1992. Nanosystems: Molecular Machinery, Manufacturing, and Computation. Wiley, New York. Dubensky, T.W., Liu, M.A., Ulmer, J.B., 2000. Delivery systems for gene-based vaccines. Mol. Med. 6, 723 732. Eng, T.L., 1999. On solving 3CNF-satisability with an in vivo algorithm. BioSystems 51, 135 141. Erbacher, P., Zou, S., Bettinger, T., Steffan, A.M., Remy, J.S., 1998. Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. Pharm. Res. 15, 1332 1339. Fahy, G.M., 1993a. Molecular nanotechnology. Clin. Chem. 39, 2011 2016. Fahy, G.M., 1993b. Molecular nanotechnology and its possible pharmaceutical implications. In: Bezold, C., Halperin, J.A., Eng, J.L. (Eds.), 2020 Visions: Health Care Information Standards and Technologies. US Pharmacopoeia Convention, Rockville, MD, pp. 152 159. Fattal, E., Rojas, J., Youssef, M., Couvreur, P., Andremont, A., 1991. Liposome-entrappted ampicilin in the treatment of experimental murine listeriosis and salmonellosis. Antimicrob. Agents Chemother. 35, 770 772. Fattal, E., Vauthier, C., Aynie, I., Nakada, Y., Lambert, G., Malvy, C., Couvreur, P., 1998. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J. Control. Release 53, 137 143. Faulhammer, D., Cukras, A.R., Lipton, R.J., Landweber, L.F., 2000. Molecular computation: RNA solutions to chess problems. Proc. Natl. Acad. Sci. USA 97, 1385 1389. Freitas, R.A., 1996a. The future of computers. Analog 116, 57 73. Freitas, R.A., 1996b. Respirocytes: high performance articial nanotechnology red blood cell. Nanotechnol. Magazine 2, 8 13. Freitas, R.A., 1998. Exploratory Design in Medical Nanotechnology: A Mechanical Articial Red Cell, Artif. Cells, Blood Subtit. and Immobil. Biotech, 26, 411 430. (see also http://www.foresight.org/Nanomedicine/Respirocyte).

136

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138 Kyoda, K., Kitano, H., 1999. Simulation of genetic interaction for Drosophila leg formation. Pac. Symp. Biocomput. 77 89. Lagally, E.T., Simpson, P.C., Mathies, R.A., 2000. Monolithic integrated microuid DNA amplication and capillary electrophoresis analysis system. Sens. Actuat. 63, 138 146. Lambert, G., Fattal, E., Couvreur, P., 2001. Nanoparticulate systems for the delivery of antisense oligonucleotides. Adv. Drug. Deliv. Rev. 47, 99 112. Lampton, C., 1995. Nanotechnology promises to revolutionize the diagnosis and treatment of diseases. Genetic Eng. News 4, 23. Langer, R., 2000. Biomaterials in drug delivery and tissue engineering: one laboratorys experience. Acc. Chem. Res. 33, 94 101. Leon, K., Perez, R., Lage, A., Carneiro, J., 2001. Three-cell interactions in T cell-mediated suppression? A mathematical analysis of its quantitative implications. J. Immunol. 166, 5356 5365. Levine, H.A., Pamuk, S., Sleeman, B.D., Nilsen-Hamilton, M., 2001. Mathematical modeling of capillary formation and development in tumor angiogenesis: penetration into the stroma. Bull. Math. Biol. 63, 801 863. Liaw, J., Aoyagi, T., Kataoka, K., Sakurai, Y., Okano, T., 1998. Visualization of PEO-PBLA-pyrene polymeric micelles by atomic force microscopy. Pharm. Res. 11, 1721 1726. Linial, M., Linial, N., 1995. On the potential of molecular computing. Science 268, 481. Lipton, R.J., 1995. DNA solution of hard computational problems. Science 268, 542 545. Liu, Q., Wang, L., Frutos, A.G., Condon, A.E., Corn, R.M., Smith, L.M., 2000. DNA computing on surfaces. Nature 403, 175 179. Lo, Y.M., Yiu, K.F., Wong, S.L., 1995. On the potential of molecular computing. Science 268, 481 482. Loke, S.L., Stein, C.A., Zang, X.H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J.S., Neckers, L.M., 1989. Characterization of oligonucleotide transport into living cells. Proc. Natl. Acad. Sci. USA 86, 3474 3478. Maki, Y., Tominaga, D., Okamoto, M., Watanabe, S., Eguchi, Y., 2001. Development of a system for the inference of large scale genetic networks. Pac. Symp. Biocomput. 446 458. Mao, C., Sun, W., Shen, Z., Seeman, N.C., 1999. A nanomechanical device based on the B Z transition of DNA. Nature 397, 144 146. Matsuno, K., Yamamoto, M., Shiba, T., Ohuchi, A., 2001. An in vivo approach to molecular computation. Proceedings of the Sixth International Symposium on Articial Life and Robotics (AROB 6th 01), Tokyo, Japan, pp. 532 535. Merkle, R.C., 1996. Nanotechnology and medicine. In: Klatz, R.M. (Eds.), Advances in Anti-Aging Medicine. Liebert Press, pp. 277 286. (see also http://nano.xerox.com/ nanotech/nanotechAndMedicine.html).

Gardner, T.S., Cantor, C.R., Collins, J.J., 2000. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339 342. Gewirtz, A.M., 1999. Myb targeted therapeutics for the treatment of human malignancies. Oncogene 18, 3056 3062. Guarnieri, F., Fliss, M., Bancroft, C., 1996. Making DNA add. Science 273, 220 223. Guetens, G., van Cauwenberghe, K., de Boeck, G., Maes, R., Tjaden, U.R., van der Greef, J., Highley, M., van Oosterom, A.T., de Bruijn, E.A., 2000. Nanotechnology in bio/clinical analysis. J. Chromatogr. B. Biomed. Sci. Appl. 739, 139 150. Hamahashi, S., Kitano, H., 1998. Stimulation of Drosophila embryogenesis. In: Adami, C., Belew, R.K., Kitano, H., Taylor, C.E. (Eds.), Articial Life VI. Proceedings of the Sixth International Conference on Articial Life. MIT Press, Cambridge, MA, pp. 151 160. Head, T., Rozenberg, G., Bladergroen, R.S., Breek, C.K., Lommerse, P.H., Spaink, H.P., 2000. Computing with DNA by operating on plasmids. BioSystems 57, 87 93. Illum, L., Jabbal-Gill, I., Hinchcliffe, M., Fisher, A.N., Davis, S.S., 2001. Chitosan a novel nasal delivery system for vaccines. Adv. Drug. Deliv. Rev. 51, 81 96. Jenning, V., Gysler, A., Schafer-Korting, M., Gohla, S.H., 2000. Vitamin A loaded solid lipid nanoparticles for topical use: occlusive properties and drug targeting to the upper skin. Eur. J. Pharm. Biopharm. 49, 208 211. Kay, M.A., Manno, C.S., Ragni, M.V., Larson, P.J., Couto, L.B., McClelland, A., Glader, B., Chew, A.J., Tai, S.J., Herzog, R.W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A.W., High, K.A., 2000. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 3, 257 261. Kellermayer, M.S., Smith, S., Bustamante, C., Granzier, H.L., 2000. Mechanical manipulation of single titin molecules with laser tweezers. Adv. Exp. Med. Biol. 481, 111 126. Knight, C.G., 1981. Liposomes from physical structure to therapeutic applications. Elsevier, Amsterdam. Kozusko, F., Chen, P., Grant, S.G., Day, B.W., Panetta, J.C., 2001. A mathematical model of in vitro cancer cell growth and treatment with the antimitotic agent curacin A. Math. Biosci. 170, 1 16. Kreuter, J., 2001. Nanoparticulate systems for brain delivery of drugs. Adv. Drug. Deliv. Rev. 47, 65 81. Kubik, T., Bogunia-Kubik, K., 2002. Computationally complex problems resolved by calculations at the DNA level. Biotechnologia 1, in press. Kubik, T., Bogunia-Kubik, K., Sugisaka, M., 2001. Graph coloring problem solving by calculations at the DNA level with operating on plasmids. Proceedings of the International Conference on Control, Automation and Systems (2001 ICCAS) Jeju, Korea, pp. 433 436. Kumar, M.N.V., 2000. Nano and microparticles as controlled drug delivery devices. J. Pharm. Pharmaceut. Sci. 3, 234 258.

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138 Mountain, A., 2000. Gene therapy: the rst decade. Trends Biotechnol. 18, 119 127. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., Erlich, H., 1986. Specic enzymatic amplicatiopn of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263 273. Northrup, M.A., Benett, B., Hadley, D., Landre, P., Lehew, S., Richerd, J., Stratton, P., 1998. A miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers. Anal. Chem. 70, 918 922. Onodera, M., Ariga, T., Kawamura, N., Kobayashi, I., Ohtsu, M., Yamada, M., Tame, A., Furuta, H., Okano, M., Kotani, H., Mc Garrity, G.J., Blease, R.M., Sakiyama, Y., 1998. Successful peripheral T-lymphocyte-direct gene transfer for a patient with severe combine immune deciency caused by adenosine deaminase deciency. Blood 91, 30 36. Ouyang, Q., Kaplan, P.D., Liu, S., Libchaber, A., 1997. DNA solution of the maximal clique problem. Science 278, 446 449. Permiakov, N.K., Ananian, M.A., Sorokovo, V.I., Luskinovich, P.N., 1998. Scanning probe microscopy and medico-biological nanotechnology: history and prospects. Arkh. Patol. 60, 9 13. Factor VIII Transkaryotic Therapy Study Group, Roth, D.A., Tawa, N.E., OBrien, J.M., Treco, D.A., Selden, R.F., 2001. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia. A. New Engl. J. Med. 344, 1735 1742. Rozen, D.E., McGrew, S., Ellington, A.D., 1996. Molecular computing: does DNA compute. Curr. Biol. 6, 254 257. Rudin, C.M., Holmlund, J., Fleming, G.F., Mani, S., Stadler, W.M., Schumm, P., Monia, B.P., Johnston, J.F., Geary, R., Yu, R.Z., Kwoh, T.J., Dorr, F.A., Ratain, M.J., 2001. Phase I Trial of ISIS 5132, an antisense oligonucleotide inhibitor of c-raf-1, administered by 24-hour weekly infusion to patients with advanced cancer. Clin. Cancer Res. 7, 1214 1220. Russell-Jones, G.J., 1998. Use of vitamin B12 conjugates to deliver protein drugs by the oral route. Crit. Rev. Ther. Drug Carrier Syst. 15, 557 586. Russell-Jones, G.J., Alpers, D.H., 1999. Vitamin B12 transporters. Pharm. Biotechnol. 12, 493 520. Russell-Jones, G.J., Arthur, L., Walker, H., 1999. Vitamin B12-mediated transport of nanoparticles across Caco-2 cells. Int. J. Pharm. 179, 247 255. Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B., Erlich, H.A., 1986. Analysis of enzymatically amplied B-globin and HLADQa DNA with allele-specic oligonucleotide probes. Nature 324, 163 166. Sambrook, J., Frich, E.F., Maniatis, T., 1989. Gel electrophoresis of DNA. In: Molecular Cloning. A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press Sections 6.2 6.8. Schroeder, U., Sommerfeld, P., Ulrich, S., Sabel, B.A., 1998. Nanoparticle technology for delivery of drugs across the blood brain barrier. J. Pharm. Sci. 87, 1305 1307.

137

Seeman, N.C., 1999. DNA engineering and its application to nanotechnology. Trends Biotechnol. 17, 437 443. Seeman, N.C., Wang, H., Yang, X., Liu, F., Mao, C., Sun, W., Wenzler, L., Shen, Z., Sha, R., Yan, H., Wong, M.H., Sa-Ardyen, P., Liu, B., Qiu, H., Li, X., 1998. New motifs in DNA nanotechnology. Nanotechnology 9, 257 273. Shi, Z., Curiel, D.T., Tang, D.C., 1999. DNA-based non-invasive vaccination onto the skin. Vaccine 17, 2136 2141. Smolen, P., Baxter, D.A., Byrne, J.H., 2000. Mathematical modeling of gene networks. Neuron 26, 567 580. Smolen, P., Baxter, D.A., Byrne, J.H., 2000a. Modeling transcriptional control in gene networks methods, recent results, and future directions. Bull. Math. Biol. 62, 247 292. Soppimath, K.S., Aminabhavi, T.M., Kulkarni, A.R., Rudzinski, W.E., 2001. Biodegradable polymeric nanoparticles as drug delivery devices. J. Control. Release 70, 1 20. Soukup, G.A., Breaker, R.R., 1999. Nucleic acids molecular switches. Trends Biotechnol. 17, 469 476. Stella, B., Arpicco, S., Peracchia, M.T., Desmaele, D., Hoebeke, J., Renoir, M., De Angelo, J., Cattel, L., Couvreur, P., 2000. Design of folic acid-conjugated nanoparticles for drug targeting. J. Pharm. Sci. 89, 1452 1464. Swanson, K.R., True, L.D., Lin, D.W., Buhler, K.R., Vessella, R., 2001. A quantitative model for the dynamics of serum prostate-specic antigen as a marker for cancerous growth: an explanation for a medical anomaly. Am. J. Pathol. 158, 2195 2199. Tarakanov, A., Dasgupta, D., 2000. A formal model of an articial immune system. BioSystems 55, 151 158. Taton, T.A., 2001. Boning up on biology. Nature 412, 491 492. Taylor, T.B., Harvey, S.E., Albin, M., Lebak, L., Ning, Y., Mowat, I., Schuerlein, T., Principe, E., 1998. Process control for optimal PCR performance in glass microstructures. Biomed. Microdevices 1, 65 70. Tomita, M., Hashimoto, K., Takahashi, K., Shimizu, T.S., Matsuzaki, Y., Miyoshi, F., Saito, K., Tanida, S., Yugi, K., Venter, J.C., Hutchison, C.A., 1999. E-CELL: software environment for whole-cell simulation. Bioinformatics 15, 72 84. (see also: http://www.e-cell.org). Triggle, D.J., 1999. Rho Chi lecture. Pharmaceutical sciences in the next millennium. Ann. Pharmacother. 33, 241 246. Ulmer, J.B., Sadoff, J.C., Liu, M.A., 1996. DNA vaccines. Curr. Opin. Immunol. 8, 531 536. van Noort, D., McCaskill, S., 2001. Optically programmable computation with DNA molecules. Proceedings of the Sixth International Symposium on Articial Life and Robotics (AROB 6th 01), Tokyo, Japan, pp. 393 396. Vohradsky, J., 2001. Neural network model of gene expression. FASEB J. 15, 846 854. Wasiewicz, P., Rudnicki, R., Mulawka, J.J., Lesyng, B., 2000. Ading numbers with DNA. Proceedings of the 2000 IEEE International Conference on Systems, Man & CyberneticsSMC2000, Nashville, USA, pp. 265 270. Waters, J.S., Webb, A., Cunningham, D., Clarke, P.A., Raynaud, F., di Stefano, F., Cotter, F.E., 2000. Phase I clinical and pharmacokinetic study of bcl-2 antisense oligonucle-

138

K. Bogunia -Kubik, M. Sugisaka / BioSystems 65 (2002) 123 138 Wooley, A.T., Guillemette, C., Cheung, C.L., Housman, D.E., Lieber, C.M., 2000. Direct haplotyping of kilobase-size DNA using carbon nanotube probes. Nat. Biotechnol. 18, 760 763. Yakubov, L.A., Deeva, E.A., Zarytova, V.F., Ivanova, E.M., Ryte, L.V., Vlassov, V.V., 1989. Mechanism of oligonucleotides uptake vy cells: involvement of specic receptors. Proc. Natl. Acad. Sci. USA 86, 6454 6458. Yates, A., Bergmann, C., van Hemmen, J.L., Stark, J., Callard, R., 2000. Cytokine-modulated regulation of helper T cell populations. J. Theor. Biol. 206, 539 560. Zanders, E.D., 2000. Gene expression analysis as an aid to the identication of drug targets. Pharmacogenomics 1, 375 384. Zhang, N.Y., Tan, H.D., Yeung, E.S., 1998. Automated and integrated system for high-throughput DNA genotyping directly from blood. Anal. Chem. 71, 1138 1145.

otide therapy in patients with non-Hodgkins lymphoma. J. Clin. Oncol. 18, 1812 1823. Waters, L.C., Jacobson, S.C., Kroutchinina, N., Khandurina, J., Foote, R.S., Ramsey, J.M., 1998. Multiple sample PCR amplication and electrophoretic analysis on a microchip. Anal. Chem. 70, 5172 5176. Wilding, P., Kricka, L.J., Cheng, J., Hvichia, G., Shoffner, M.A., Fortina, P., 1998. Integrated cell isolation and polymerase chain reaction analysis using silicon microlter chambers. Anal. Biochem. 257, 95 100. Wooley, A.T., 2001. Biomedical microdevices and nanotechnology. Trends Biotechnol. 19, 38 39. Wooley, A.T., Hadley, D., Landre, P., de Mello, A.J., Mathies, R.A., Northrup, M.A., 1996. Functional integration of PCR amplication and capillary electrophoresis in a microfabricated DNA analysis device. Anal. Chem. 68, 4081 4086.