nano

BioSystems 65 (2002) 123–138

From molecular biology to nanotechnology andnanomedicine

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

a Faculty of Engineering, Oita Uni�ersity, Dannoharu 700, 870-1192 Oita, Japanb L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12,

53-114 Wroclaw, Poland

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 thestructure 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-basedcomputational approaches. On the other hand, people have started to be more interested in mimicking real life, andmodeling the structures and organisms that already exist in nature for the further evaluation and insight into theirbehavior and evolution. These factors, among others, have led to the description of artificial organelles or cells, andthe construction of nanoscale devices. These nanomachines and nanoobjects might soon find a practical implementa-tion, especially in the field of medical research and diagnostics. The paper presents some examples, illustrating theprogress in multidisciplinary research in the nanoscale area. It is focused especially on immunogenetics-related aspectsand the wide usage of DNA molecules in various fields of science. In addition, some proposals for nanoparticles andnanoscale tools and their applications in medicine are reviewed and discussed. © 2002 Elsevier Science Ireland Ltd.All rights reserved.

Keywords: DNA analyses; DNA computation; Artificial cells modeling; Nanoparticles; Nanomedicine

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1. Nanotechnology—operations on DNAmolecules

The discovery of polymerase chain reaction(PCR) (Mullis et al., 1986; Saiki et al., 1986)opened a new area of biological research. The

impact can be observed not only by the greatprogress in the field of molecular biology, but alsoin many achievements in other related fields ofscience. Molecular biology techniques have beenimplemented successfully in biology, biotechnol-ogy, medical science, diagnostics, and many more.The introduction of PCR resulted in improvingthe old, and designing the new laboratory devicesfor PCR amplification and analysis of amplifieddeoxyribonucleic acid (DNA) fragments. In paral-

* Corresponding author. Tel./fax: +81-975-54-7841.E-mail address: [email protected] (K. Bogunia-

Kubik).

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K. Bogunia-Kubik, M. Sugisaka / BioSystems 65 (2002) 123–138124

lel to these efforts, the nature of DNA molecules,their construction, and their potential use as acomputation medium have attracted many re-searchers. In addition, some studies concerningmimicking living systems, as well as developingand constructing artificial nanodevices, such asbiomolecular sensors and artificial cells, have beenconducted. These factors, among others, are theorigins of a new nanoscale technology domain.

1.1. Technical tools for micro- and nanoscaleDNA analysis

Most research, especially in biological sciences,cannot be conducted without specialized labora-tory tools and equipment. Progress in the scien-tific domain is accompanied, if ever possible, bythe proposals in the technical domain. Such coin-cidence can be observed between recent achieve-ments in molecular biology and in thedevelopment of equipment to perform analysesand diagnostic procedures based on DNA (orribonucleic acid (RNA)). Particular attentionshould be paid to PCR and the electrophoresisapparatus, which have been improved signifi-cantly and introduced into routine scientific labo-ratory work.

Electrophoresis apparatuses are used for iden-tification of the products of PCR amplification.They are built on the principle that in a microen-vironment with 5�pH�9, DNA molecules arecharged negatively and they are able to migrate inan electric field. When analysis is performed in agel, the dynamics of the migration depend mostlyon the DNA molecules’ size (Sambrook et al.,1989).

Originally, PCR amplifications were performedin water thermocyclers in a reaction volume of100 �l employing 30 PCR cycles for over 3 h. Butin routine laboratory work, water thermocyclershave been replaced by more modern machinesthat use Peltier elements and heating blocks.These machines allow PCR amplifications to beperformed in 200 �l tubes in a final reactionvolume of 10 �l using 30 PCR cycles for about 1.5h (i.e. PTC-200 DNA Engine Line Gradient Fea-ture, MJ Research Inc., Waltham, MA, USA).Currently, even more precise devices are available.

One of the first microdevices to both amplifyand detect PCR products without intermediatesteps was reported in Wooley et al. (1996). Thisdevice, composed of a silicon reaction chamberattached to a glass capillary electrophoresis sepa-ration channel, had a 10 �l volume and performedamplifications in 15 min. Since then, a great num-ber of different microfluidic PCR devices havebeen fabricated in silicon (Chuadhari et al., 1998;Daniel et al., 1998; Northrup et al., 1998; Wildinget al., 1998), glass (Taylor et al., 1998; Waters etal., 1998), silicon–glass hybrids (Cheng et al.,1996), and in fused silica capillaries (Zhang et al.,1998).

During the BioMENS and Biomedical Nano-technology World 2000 conference (reviewed byWooley (2001)) many interesting machines forbiochemical analysis were presented. These in-cluded mainly micromachines allowing differentDNA analyses to be performed, e.g. PCR amplifi-cation, electrophoresis, and sequencing. One ofthem was a high-throughput biochemical mi-croprocessor is able to perform rapid and parallelDNA sequencing on 96 samples to 500 bases,corresponding to a throughput of �100 kb/h. Anintegrated device for PCR amplification and elec-tophoretic analysis was also interesting. This wasa fully integrated system in glass for the manipu-lation, amplification, and capillary electrophoresisseparation of submicroliter volumes of DNA (La-gally et al., 2000). In this system, the use of alow-volume reactor with 280 nl PCR chambersand thin-film heaters permitted thermal cycletimes as fast as 30 s. The amplified product waslabeled with an intercalating fluorescent dye andinjected directly into a microfabricated capillaryelectrophoresis system. The reaction could startwith as few as 20 DNA template copies permicroliter (5 to 6 per chamber).

During the same conference, miniaturized high-speed thermal cycling chambers were shown.These chambers were able to complete 10 PCRcycles in 30 s and successfully amplify a DNAsample in fewer than 4 min. Moreover, a methodfor the multiplex detection of polymorphic sitesand direct determination of haplotypes in 10-kbDNA fragments, using single-walled carbon nan-otube atomic force microscopy probes, was also

K. Bogunia-Kubik, M. Sugisaka / BioSystems 65 (2002) 123–138 125

described (Wooley et al., 2000). This techniquewas based on the hybridization of labeledoligonucleotides to complementary target DNAsequences. It allowed the direct determination ofhaplotypes in patient samples and had been em-ployed for study on UGT1A7, a cancer risk-fac-tor gene (Wooley et al., 2000). In addition, anovel method for electrochemical detection ofDNA hybridization with the use of conductivehydrogels was proposed.

These micro- and nanotechnological tools arebeing used to sequence genomes and diagnosediseases. The analyzers mentioned above may al-low much faster, more specific, and more preciseDNA analysis and DNA-based diagnostic proce-dures than used presently. The reduction of sam-ple volume, like the amount of necessary DNAtemplate, is an additional advantage, especially inthe case of a limited availability of samples (e.g.for pediatric patients). Employing more efficientmachines may also improve and shorten the timeof analyses, which can be of particular impor-tance, e.g. when searching for suitable donors fortransplantation for patients suffering from hema-tological disorders (Bogunia-Kubik and Lange,2000), where the time factor is critical.

1.2. Computation at DNA le�el

In recent years a great interest in biologicallyinspired systems has been seen among researchersdeveloping new computer techniques. One branchis based on the fact that genetic information isstored in nucleic acid molecules. Moreover, theseDNA molecules possess massively parallel pro-cessing capabilities and are easy to manipulate ina genetic engineering laboratory. These facts in-spired hopes that DNA-based computers, if built,might handle millions of operations in paralleland solve hard computational problems in a rea-sonable amount of time (Kubik and Bogunia-Kubik, 2002).

1.2.1. In �itro DNA computationThe principle of computing with an ordinary

computer relies on transmitting logical signalsthrough an especially designed network of paths,gates, and connections. These signals have the

form of electrical impulses, whose voltage level isinterpreted as a logical 0 or 1. Computer pro-grams are collections of words consisting of suchbinary values that, as electrical impulses, aretransmitted through the computer’s electroniccontrol devices.

DNA-based computation has a quite differentnature. In the case of DNA, words are composedof specific sequences of DNA floating in amedium (there are no conductor paths), and theprocess of computing has the form of chemicalreactions. With a DNA computer, computationrequires synthesizing particular sequences ofDNA in a separate process (according to theproblem) and letting them react in ‘a test tube’(reactions are performed extracellularly in solu-tion or affixed to a surface of glass or silicon).These kinds of DNA-based computational ap-proaches have been called in vitro DNAcomputation.

The practical possibility of using in vitro DNAcomputation was demonstrated by Adelman in hispioneering work (Adelman, 1994). He used DNA-based computation to solve a seven-node Hamil-tonian path problem (HPP). The HPP is definedas follows: given a graph, is there a path throughthe 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, sothe experiment performed by Adelman must beregarded as an illustration of the potential ofDNA computing. At first, Adelman constructedtwo sets of 20-nucleotide long sequences repre-senting particular vertices and particular connec-tions between two vertices in a 7-node graph,respectively. In each vertex representation, tenfirst and ten last nucleotides were unique and wereinterpreted as sequences representing the ‘input’and ‘output’ of the vertex, respectively. Sequencesrepresenting connections were constructed fromthe sequences that were complementary to the‘outputs’ and ‘inputs’ of the adequate vertices.

Adelman then started the ‘calculation’ part ofthe experiment. He used PCR to amplify all possi-ble connections/paths, which began with vertex 0and ended with vertex 6. Next, the electrophoresisof amplification products led him to the separa-tion of sequences with length corresponding to the


path that passed through all vertices. Furthersteps of the analysis allowed the selection of thosesequences, which represented paths without re-peated vertices. This part of the experimentneeded serial hybridizations with the use of ade-quate panels of nucleotide sequences immobilizedon magnetic beads. Any sequences that remainedafter that analysis constituted solutions of theproblem. Thus to solve the HPP, Adelman em-ployed a synthesis of DNA fragments, amplifica-tion with the use of PCR, electrophoresis,probing, and finally sequencing the remainingDNA fragments selected throughout this multi-step analysis.

Following in the path of Adelman’s experiment,Lipton (1995) proposed a series of experiments totackle NP-complete satisfaction problems (SAT).The SAT problem is defined as follows: given aBoolean formula F, is there an assignment ofvalues to the variables of F so that F is true?Recently, Liu et al. (2000) published their workon DNA computing on surfaces. They proposed amethod of solving the NP-complete SAT problemby attaching candidate DNA oligonucleotides to asupport, and screening using successive hybridiza-tion operations.

The success of experiments using DNA frag-ments to solve calculation problems has generatedmuch more interest in the scientific world aboutthe potential properties and usage of DNAmolecules. Seeman and co-workers (Seeman,1999; Seeman et al., 1998) performed studies onDNA molecules analyzing, among other things,their secondary and tertiary structure and creatingspecific topologies, shapes, and arrangements ofligated DNA strands. They also proposed theutilization of DNA for a practical solution ofcumulative XOR calculations. For this purpose,they used appropriately constructed DNA frag-ments and employed a number of molecular biol-ogy techniques, including ligation, PCR, digestionof PCR products with restriction enzymes, andelectrophoresis.

The next examples of a practical DNA imple-mentation were demonstrated by Ouyang et al.(1997) and van Noort and McCaskill (2001). Inboth cases, the objective was to solve the NP-

complete maximum clique problem (MCP). TheMPC is defined as follows: given an undirectedgraph, find the largest set (maximum clique) of itsmutually adjacent vertices. The method ofOuyang and co-workers (Ouyang et al., 1997) wasbased on construction of 64 DNA double-stranded molecules that were analyzed by selectivedigestion with restriction enzymes. To solve theMCP, van Noort and McCaskill (2001) con-structed not only a set of appropriate DNA se-quences, but also an entire microflow reactor,where particular hybridization reactions of ana-lyzed DNA strands with ‘selecting’ DNA se-quences immobilized on the paramagnetic beadsoccurred. Each analyzed sequence offered a bi-nary encoding of particular subgroups of nodes inthe graph considered, where 0 (or 1) was repre-sented by the whole fragment of a DNA sequence(not only by a single nucleotide base pair). Theend effect of this selection (the solution) wasobserved by CCD cameras and a microscope.

Some other proposals of DNA computationhave been based on the usage of specifically de-signed circular DNA molecules (DNA plasmids)as a computational tool. These methods wereused, for example, for analysis of the NP-com-plete algorithmic problem of computing the cardi-nal number of a maximal independent subset(MIS) of a graph (Head et al., 2000) and solvingthe graph coloring problem (GCP) (Kubik et al.,2001). In both studies, plasmids contained spe-cially inserted DNA sequences so that each onewas bordered by a characteristic pair of restrictionenzyme sites. The ‘calculation’ steps involved em-ploying of a number of restriction endonucleasetreatments to the computational plasmid.

In the case of the MIS problem, the ‘calcula-tions’ ended with ‘a tube’ containing plasmidsrepresenting all independent subsets of the graph(subsets of vertices not connected by the edge). Asolution to GCP (assignments of colors to graphvertices such that no two vertices connected by anedge are painted with the same color while thetotal number of colors is minimized) was foundby extending this procedure with some additionalrestriction enzyme treatments and serial hy-bridization steps (Kubik et al., 2001).


1.2.2. In �i�o DNA computationDNA computation as described by the pioneer

work of Adelman and many followers is currentlycalled in vitro DNA computation. But researchershave also tried to find another method for DNA-based computation. The idea is to use the proper-ties of living cells and organisms, more exactly, touse DNA molecules and solve computationalproblems within a living cell. In this way, in vivoapproaches to DNA computation have beenintroduced.

In one study, Matsuno et al. (2001) proposedan in vivo method for computation of two logicfunctions: T(X)=X, and F(X)= �X. Theydemonstrated the method using the biologicalproperties of Escherichia coli bacteria, and theirability to synthesize �-galactosidase (controlled bylac-operon) in the presence of an induction factor(IPTG). So, two biological processes, gene expres-sion and protein synthesis, were involved in thecomputation process (Matsuno et al., 2001).

There are also other reports pointing out thepossibility of in vivo computation. For example,Gardner et al. (2000) presented another computa-tional approach in E. coli that proposed the con-struction of a genetic toggle switch forming acellular memory unit. Eng (1999) proposed solv-ing the 3CNF-SAT problem (a special case ofSAT problem) with an in vivo algorithm. Otherinteresting in vivo DNA computation proposalson constructing DNA or RNA/rybozyme basedmolecular switches come from the study of Gard-ner et al. (2000) and Soukup and Breaker (1999).

1.2.3. Contrary �iews on molecular computationThe implementation of in vitro and in vivo

molecular computation (recently based also onRNA (Cukras et al., 1999; Faulhammer et al.,2000)) illustrates how knowledge coming frommolecular biology and genetics can be imple-mented to solve hard computational problems.Although the practical benefits of DNA-basedcomputational schemes are still questionable andthe vast majority of work to date has been theo-retical, there have been many allusions to poten-tial uses of this emerging computationalparadigm. It has been suggested that DNA/biomolecular computation is faster, uses less en-

ergy, has a greater potential for informationstorage, and the ability to handle much largernumbers of operations in parallel than conven-tional computing (Baum, 1995; Chen and Wood,2000). According to Baum (1995), the memory ofmolecular computers may be of greater capacitythan that of a human brain.

Although the technical potential of DNA-basedcomputation looks very promising, the practicalusage, including the high costs operations per-formed, seems to be limited. DNA-based calcula-tions require a large number of specificallyencoded nucleotides (Bunow, 1995). It has beencalculated that a HPP with 23 nodes would needkilogram quantities of DNA (Lo et al., 1995), butthe amount of DNA required to solve a 70-nodeproblem would equal over 1000 kg of nucleicacids (Linial and Linial, 1995). In addition, DNAcomputation might be erroneous and might causetechnical problems as has been investigated pre-liminarily (Aoi et al., 1999) by analyzing theerrors that may occur during the ligation processof DNA sequences, and discussed by Cox et al.(1999).

Moreover, to date, no large computationallycomplex problem has been encoded in DNA andsolved by molecular biology (Rozen et al., 1996).Researchers in this field look for problems thatcould be solved by molecular computers ratherthan at the method of analysis. DNA computa-tion is based generally on the generation of allpossible DNA-encoded candidate answers fol-lowed by a good candidate search and finally theinterpretation of the results, for example, by can-didate sequencing. This technique has not beenused strictly to solve arithmetic problems, butrather to help to find a solution or a set ofsolutions that meet specific criteria from the poolof possible combinations. However, there hasbeen an attempt to use DNA to sum binaryinteger numbers (Guarnieri et al., 1996;Wasiewicz et al., 2000).

Thus, performing calculations employingbiomolecules and using genetic engineering tech-nology, although now questionable, may soonbecome the most widely used tool forcomputation.


1.3. Modeling of life—artificial cells

We have indicated that biological tools (DNAmolecules, enzymes, and different kind of biologi-cal reactions), with some limitation, can be usedto solve computational problems. In this way,biology helps mathematical sciences. However,even when employing very modern technical toolsand high-resolution devices, it is not possible toobserve in great details the processes ongoingwithin living cells or look deeply at and analyzethe mechanisms of cell–cell interactions. For thatreason, mathematical tools and computer soft-ware can be employed, for the description andmodeling of structures and organisms already ex-isting in nature. This theoretical research mayhelp in the evaluation and give some insight intothe behavior and evolution of biological systems.A few examples of mathematical models, includ-ing the simulation of gene expression, proteinproduction, and interactions of the cells, will bepresented below.

1.3.1. Models of li�ing organismsOn the basis of a bacteria genome, Tomita et

al. (1999) constructed a model of a hypotheticalcell with only 127 genes being sufficient for tran-scription, translation, energy production, andphospholipid synthesis. Most of the genes weretaken from Mycoplasma genitalium, the organismhaving the smallest-known chromosome, whosecomplete 580 kb genome sequence was deter-mined in 1995. They developed the E-cell system,which is a computer software environment formodeling and simulation of the cell, in order tounderstand how all the cellular proteins workcollectively as a living cell system. The goal of thisstudy is, by attempting to understand the dynam-ics in living cells, to predict consequences ofchanges introduced into the cell and/or its envi-ronment, e.g. knocking out genes or alteringavailable metabolites.

It is also worth noting work on a more complexorganism, the fruit fly Drosophila, for simulationof Drosophila embryogenesis (Hamahashi and Ki-tano, 1998) or leg formation (Kyoda and Kitano,1999), as examples of simulations of gene interac-tions and expression.

1.3.2. Mathematical modeling of gene networksDNA duplication (DNA synthesis), transcrip-

tion (synthesis of mRNA on the DNA template),and translation (protein production) are processesthat take place in any living cell. A number ofmodels have been proposed in order to analyzeand simulate interactions between genes (gene net-works) and the control of gene transcription andexpression processes gaining insights into thestatic and dynamic behavior of complex biologicalsystems. These, for example, have been shownand reviewed by Smolen and co-workers (Smolenet al., 2000, 2000a). The regulatory effect of onegene product on the expression of other geneswere analyzed by Vohradsky (2001). Moreover,algorithms for inference of genetic networks(called AIGNET systems), introduced by Maki etal. (2001), were implemented for analysis of theinterference of a genetic network composed fromjust 30 genes. This model allows the analysis ofthe different patterns of gene expression, includ-ing some kinds of gene perturbations, such asdisruption or over expression. In addition, thesekinds of analyses may lead to the identification ofDNA sequences that may serve as potentialtargets of antisense oligonucleotides and se-quences of genes in, discussed later, antisense andgene therapies (Zanders, 2000).

1.3.3. Mathematical model of an immune systemCell interactions and co-operations constitute

the bases of many mathematical simulations.Studies have been undertaken to describe andanalyze the relations between T and B cells of theimmune system that will be described here inmore detail.

B and T cells are two major classes oflymphocytes playing an important role in theimmune system response. B cells, B lymphocytes,upon activation by an antigen, differentiate intocells producing antibodies. T cells are subsets oflymphocytes defined by their development in thethymus and by their heterodimeric receptors (Tcell receptor (TCR)) that can recognize the anti-gen presented by MHC (major histocompatibilitycomplex) molecules. MHC molecules are ex-pressed on antigen presenting cells (such asmacrophage, B lymphocytes or dendritic cells).


Two classes of MHC molecules present antigenicpeptides to T lymphocytes. MHC class Imolecules present peptides generated in cytosol toCD8 T cells, and MHC class II molecules presentpeptides degradated in cellular vesicles to CD4 Tcells. CD4 T cells, T helper cells, can help B cellsmake antibodies in response to an antigenic chal-lenge. The other kind of T lymphocytes, cytotoxicT cells, which are mostly CD8 T cells, can killother cells. This is important in host defenseagainst cytosolic pathogens. Th1 and Th2 cells,and Tc1 and Tc2 cells, are the next sets of Th andTc cells, respectively. The Th1 (inflammatoryCD4 cells) subtype associates with cell-mediatedimmunity, while the Th2 (helper CD4 cells) typepromotes B cell growth and antibody production.

Agrawal and Linderman (1996) employedmathematical modeling to investigate the effect ofantigen processing and presentation on the Th cellresponse. Using two mathematical models, theygenerated theoretical curves in order: (i) to relatethe external antigen concentration to the numberof MHC–peptide complexes present on APCs,and (ii) to relate the number of MHC–peptidecomplexes on the APC to the number of boundTCRs on the Th cell. Our model showed thatmodifications to peptide antigen resulted in al-tered MHC–peptide and/or TCR/MHC–peptideaffinities and the associated kinetic bindings. Thesecond model predicted that: (i) the TCR/MHC–peptide affinity alone has a dramatic effect on thenumber of bound TCRs at smaller but not largeraffinity; (ii) the number of MHC–peptide com-plexes required to obtain a particular number ofbound TCRs varies over a range, depending onthe TCR–MHC affinity; and (iii) that a largevalue of one affinity (e.g. a large MHC–peptideaffinity) can compensate a small value of anotherone (e.g. a small TCR/MHC–peptide affinity).

In the same year, Carneiro et al. (1996) de-scribed a model of the immune network with B–Tcell co-operation, where B cell activation wasdependent on T cell help, and activated T cellswere downregulated by engagement of their TCRsby anti-TCR soluble immunoglobulines. Theytested their model by examining several proto-typic situations with a small number of T and Bcell clones. (A clone is a population of cells

derived from a common progenitor.) One of thelatest reports from this group describes anotherproposal of computer simulation used for analysisof interactions between regulatory and target Tcells, using the mechanism of T cell mediatedsuppression (Leon et al., 2001).

Recently, Tarakanov and Dasgupta (2000) pro-posed a model of an artificial immune system,referred to as a formal immune system (FIS). Thismathematical approach is based on the features ofantigen–antibody bindings. It provides a mathe-matical description of B and T cells, with a simu-lation of B and T cell interactions and proteinbindings.

Coetano et al. (1998) defined a more precisecomputational model for the dynamics of Thelper lymphocytes subpopulations (Th1 and Th2cells) to study multiple responses and cross-regu-latory points, and T cell antigen presenting cellinteraction. In this study, the authors focusedespecially on pathways leading to T cell differenti-ation into Th1 and Th2 cells. These mechanismsplay a major role in the immune system oncogeny.T helper cells and the influence of cytokines ontheir regulation were the aim of another computa-tional model proposed by Yates et al. (2000).

Mathematical modeling of the immune systemand cell interaction allows investigation of theimmunological response in health and disease.For example, some studies have been carried outto analyze the mechanism of tumor cells interac-tion (Bach et al., 2001), tumor angiogenesis(Levine et al., 2001), and development (Kozuskoet al., 2001; Swanson et al., 2001).

2. Nanotechnology—materials, artificialstructures and devices

The word nanotechnology is used to describemany types of research where the characteristicdimensions are smaller than about 1000 nm. Thisdefinition covers a wide range of different scien-tific fields, from lithography (with line widths lessthan one micron) to nanomachines andnanorobots.

The term nanotechnology can be understood asaltering individual atoms and molecules at a pre-


cise location, either chemically or physically. Nano-technology also seeks to develop devices that canscan and to manipulate objects at near atomicscale, such as the atomic force microscope (Liawet al., 1998), the scanning tunnel microscope (Per-miakov et al., 1998), the laser force microscope,laser tweezers (Kellermayer et al., 2000), or liquidchromatography–mass spectrometry devices(Guetens et al., 2000). Nanotechnology (in otherwords molecular nanotechnology, molecular man-ufacturing) also comprises the building of nanos-tructures and manufacturing nanometer-scaleobjects. Therefore, DNA computation (as manip-ulation of nanoscale particles), different kinds ofmicroscopy, manipulation of molecules, con-structing nanometre resolution diagnostic andanalytical devices, and nanomachines andnanorobots, can all be described by this term—nanotechnology.

To date, several nanoscale devices have beendescribed in the literature. Among them there area few composed of nucleic acids, e.g. a nanome-chanical DNA device (Mao et al., 1999) andnucleic acid molecular switches based on ry-bozyme structure (Soukup and Breaker, 1999).Some interesting proposals have been derivedfrom Drexler (1981, 1986, 1992) and his followers(such as Merkle (1996) and Freitas, see the refer-ences below) working in the field of molecularnanotechnology and nanorobot construction.They have described nanomachines that couldfind practical implementation in medicine. One ofthem is a model of an artificial red blood cell(Freitas, 1996b, 1998).

2.1. Mechanical artificial red cells

The artificial mechanical red blood cell, calledrespirocyte, was designed by Freitas (1996a,1998). This was the first detailed design study of aspecific medical nanodevice (of the kind proposedby Dexler in ‘Nanosystems’). The proposedrespirocyte measures about 1 �m in diameter andjust flows along the bloodstream. It is a sphericalnanorobot made of 18 billion atoms. The respiro-cyte is equipped with a variety of chemical, ther-mal, and pressure sensors and an onboardnanocomputer. This device is intended to function

as an artificial erythrocyte, duplicating the oxygenand carbon dioxide transport functions of redcells, mimicking the action of naturalhemoglobin-filled red blood cells. It is expected tobe capable of delivering 236 times more oxygenper unit volume than a natural red cell. Speciallyinstalled equipment enables this device to displaymany complex responses and behaviors. Addi-tionally, it has been designed to draw power fromabundant natural serum glucose supplies, andthus is capable of operating intelligently and vir-tually indefinitely, whilst red blood cells have anatural lifespan of 4 months.

2.2. Artificial cell membranes

This is the concrete implementation of an artifi-cial cell organelle coming from Cornell and co-workers (Cornell et al., 1997). They developed asynthetic biosensor that imitates nerve cell mem-branes. This artificial membrane, an ion-basednanomachine, is composed of the following ele-ments: membrane-forming molecules that aretethered chemically to the gold surface; simple ionchannels within the membrane that facilitate thetransport of ions (like Na+, K+); a reservoirspace between the surface and the membrane tostore ions; and receptors such as antibodies at-tached to the membrane to recognize targetmolecules. The detection mechanism operates bybinding the target molecule, which alters the pop-ulation of conduction ion channel pairs within thetethered membrane. This results in a change in themembrane conduction. The analysis is based onthe measurement of changes in the membraneconduction. This is already a commercial productsupplied by the Ambri Ltd. (Chatswood, NSW,Australia) that can be used, for example, in phar-maceutical research or for rapid medical testing.

2.3. Artificial nanostructures that can interactwith and replace natural biological materials

In the report from one of the meetings ofAmerican scientists (American Chemical SocietyProSpectives, Berkeley, CA, USA, 2001) Taton(2001) presents a very intriguing proposal of anartificial bone relying on designing the synthetic


substitutes of collagen. Research on designingself-assembling, synthetic substitutes for collagenhas been conducted by a group of Stupp atNorthwestern University, Evanston, IL. Theyproposed an artificial material, composed of am-phiphilic molecules bearing a long hydrophobicalkyl group on one end and ahydrophilic peptideon the other that was able to spontaneously as-semble into cylindrical structures that resemblecollagen fibrils. Moreover, these cylinders guidedthe formation of hydroxyapatite crystallites. Whatis even more important, they formed crystallitescharacterized with orientations and sizes similarto those in natural bone. Taton emphasized thatthese observations lead to a general question:would synthesized nanomaterials be able not onlyto replicate the properties of their natural equiva-lents (cell membranes, tissues and bone marrow),but also prompt biological systems to build up onthese materials, and to produce self-assemblingstructures? Studies on such a nanostructures leadto promising materials with potential uses as im-plants and therapies. Moreover, they may some-day show how the cells interact withnanometer-sized objects in their own world.

3. Nanomedicine—another implementation ofnanotechnology

From nanotechnology it is only one step tonanomedicine, which may be defined as the moni-toring, repair, construction, and control of humanbiological systems at the molecular level, usingengineered nanodevices and nanostructures. It canalso be regarded as another implementation ofnanotechnology in the field of medical science anddiagnostics. One of the most important issues isthe proper distribution of drugs and other thera-peutic agents within the patient’s body.

3.1. Nanoparticles as carriers of therapeuticmolecules

Targeting the delivery of drugs to diseased le-sions is one of the important aspects of the drugdelivery systems. To convey a sufficient dose ofdrug to the lesion, suitable carriers of drugs are

needed. Nano- and microparticle carriers haveimportant potential applications for the adminis-tration of therapeutic molecules. Liposomes havebeen used as potential drug carriers instead ofconventional dosage forms because of theirunique advantages, which include the ability toprotect drugs from degradation, target the drug tothe site of action, and reduce the toxicity of sideeffects (Knight, 1981). However, developmentalwork on liposomes has been limited, due to inher-ent problems such as low encapsulation efficiency,rapid leakage of water-soluble drugs in the pres-ence of blood components, and poor storage sta-bility. In some cases, nanoparticles are moreefficient drug carriers than liposomes due to theirbetter stability (Fattal et al., 1991) and possessmore useful control release properties. These arethe reasons why many drugs have been associatedwith nanoparticles (e.g. antibiotics, antiviral andantiparasitic drugs, cytostatics, vitamins, proteinand peptides, including enzymes and hormones(please note the references for this paragraph ofthe paper)).

Nanoparticles are defined as being submicronic(�1 �m) colloidal systems generally made ofpolymers (biodegradable or not). They were firstdeveloped in the mid 1970s by Birrenbach andSpeiser (1976). Nanoparticles generally vary insize from 10 to 1000 nm. The drug is dissolved,entrapped, encapsulated, or attached to ananoparticle matrix. Depending upon the processused for the preparation of nanoparticles (re-viewed by Kumar, 2000; Lambert et al., 2001;Soppimath et al., 2001), nanospheres or nanocap-sules can be obtained. Nanocapsules are vesicularsystems in which the drug is confined to a cavity(an oil or aqueous core) surrounded by a uniquepolymeric membrane. Nanospheres are matrixsystems in which the drug is physically and uni-formly dispersed throughout the particles.

In recent years, biodegradable polymericnanoparticles have attracted considerable atten-tion as potential drug delivery devices, this is inview of their applications in controlling drug re-lease, their ability to target particular organs/tis-sue, as carriers of oligonucleotides in antisensetherapy, DNA in gene therapy, and in their abilityto deliver proteins, peptides and genes through


oral administration (Langer, 2000). These applica-tions of nanoparticulate systems will be discussedbelow in more detail.

3.1.1. Antisense therapyNucleic acids can be used not only to diagnose

and monitor, but also to prevent and cure dis-eases, as they constitute the bases of antisense andgene therapies. Antisense oligonucleotides haveemerged as potential gene-specific therapeuticagents and are currently undergoing evaluation inclinical trials a variety of diseases. These includeadvanced carcinoma (Cunningham et al., 2001;Rudin et al., 2001) and non-Hodgkin’s lymphoma(Waters et al., 2000; Gewirtz, 1999). Theoretically,an antisense oligonucleotide is a short fragment(15–20 bp) of deoxynucleotides characterizedwith a sequence complementary to a portion ofthe targeted mRNA. The aim of the antisensestrategy is to interface with gene expression bypreventing the translation of proteins frommRNA. There are a few mechanisms of mRNAinactivation (Lambert et al. 2001), including (i)sterical blocking of mRNA by antisense bindingand destruction antisense-mRNA hybrids byRnaseH enzyme, (ii) formation of triple helixbetween genomic double-stranded DNA andoligonucleotides, or (iii) the cleavage of targetRNA by ribozymes.

Antisense oligonucleotides are molecules thatare able to inhibit gene expression, being there-fore, potentially active for the treatment of viralinfections or cancer. However, the problems suchas the poor stability of antisense oligonucleotidesversus nuclease activity in vitro and in vivo, andtheir low intracellular penetration have limitedtheir use in therapeutics (Loke et al., 1989;Yakubov et al., 1989). In order to increase theirstability, to improve cell penetration and alsoavoid non-specific aptameric effects (leading tonon-specific binding of antisense oligonucle-otides), the use of particulate carriers such asliposomes or nanoparticles, has been considered.It has also shown interesting potentialities to bindand deliver oligonucleotides in an efficient manner(Fattal et al., 1998). Very recently, it was reportedthat antisense oligonucleotides can be encapsu-lated in nanocapsules with a size of 350�100 nm.

A formulation of these capsules might have spe-cial importance for oligonucleotide delivery. Thefirst experiments on the treatment of RAS cellsexpressing the point-mutated Ha-ras gene werepromising (Lambert et al., 2001).

In addition to the nanoparticle preparation,new techniques that enable analysis of theirproper delivery are being developed. For example,to address the crucial problem of oligonucleotidedegradation, an original assay method was pro-posed allowing, in the polyacryamide gels, quan-tification of the amount of undegradatedoligonucleotides (16 mer oligothymidilate) in tis-sues such as liver and plasma (Aynie et al., 1996).

3.1.2. Gene therapy and administration of DNA�accines

Gene therapy is a recently introduced methodfor treatment or prevention of genetic disordersbased on delivery of repaired, or the replacementof incorrect, genes. It is aimed at treating oreliminating the causes of disease, whereas mostcurrent drugs treat the symptoms. There is thewide 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 first disease approved for genetherapy treatment was adenosine deaminase(ADA) deficiency, and the first patient was treatedin 1990. Recently, Onodera et al. (1998) reportedthe use of ADA copy DNA (cDNA) for thetreatment of severe immunodeficiency (SCID) pa-tients. In addition, one of the latest reports ongene therapy demonstrates the successful treat-ment of patients with hemophilia B, with a defectin a gene encoding blood coagulation factor IX(Kay et al., 2000) and patients with hemophilia Ahaving a defect in a gene that encodes factor VIII(Roth et al., 2001). In these cases, patients’ fibrob-lasts transfected with a plasmid containing se-quences of the factor VIII gene (hemophilia Atreatment) and adeno-associated viral vectors ex-pressing human factor IX (hemophilia B) wereused for gene transfer. Application of nanotech-nological tools in human gene therapy has beenreviewed widely by Davis (1997). He describednon-viral vectors based on nanoparticles (usually


50–500 nm in size) that were already tested totransport plasmid DNA. He emphasized that nano-technology in gene therapy would be applied toreplace the currently used viral vectors by poten-tially less immunogenic nanosize gene carriers. Sodelivery of repaired genes, or the replacement ofincorrect genes, are fields where nanoscaled ob-jects could be introduced successfully.

On the other hand, genetic immunization withDNA vaccines has emerged as one of the mostpromising applications of non-viral gene therapy(Ulmer et al., 1996; Dubensky et al., 2000), havinga number of the potential advantages over con-ventional vaccines. These include: (i) the highstability of plasmid DNA, (ii) low manufacturingcosts, (iii) lack of infection risk associated attenu-ated viral vaccines, (iv) the capacity of targetmultiple antigens to one plasmid, and (v) theability to elicit both humoral and cellular immuneresponses. Until recently, intramuscular injectionwas the primary route of administration of DNAvaccines. As an alternative to intramuscular ad-ministration of plasmid DNA, researchers havebeen investigating targeting plasmid DNA to theskin using intradermal needle injection, needle-free jet injection devices, the gene gun, or recentlytopical delivery (Cui and Mumper, 2001) of for-mulated plasmid in the form of a patch, cream, orgel. The latter method may provide many advan-tages in terms of cost and patient compliance (Shiet al., 1999). Among other nanoparticles, chi-tosan, a biodegradable polysaccharide compriseof primarily D-glucosamine repeating units, hasbeen proposed by several groups as an alternativenon-viral delivery system for plasmid DNA. Se-lective chitosan polymers and chitosan oligomershave been found to efficiently condense plasmidDNA and to transfect several different cell typesin vitro and in the intestines, colon, nose, andlung (Erbacher et al., 1998; Cui and Mumper,2001).

3.1.3. Treatment of patients with peptide andprotein pharmaceuticals

Currently, this treatment is performed mostlyby injection, with accompanying patient discom-fort, increased medical costs, and reduced patientcompliance. Therefore, the systems of delivery of

peptides and proteins by the oral route haveattracted considerable attention as being mucheasier and more acceptable. Unfortunately, thisroute cannot be used with most proteins andpeptides, due to both the degradation of thesemolecules within the intestine and their poor up-take across the intestinal wall. To potentiallyovercome these problems, it has been shown thatit is possible to utilize the uptake mechanism ofvitamin B12 to enhance the oral uptake of variouspeptide and protein pharmaceuticals. In particu-lar, molecules such as luteinizing hormone releas-ing hormone (LHRH) analogues, �-interferon,erythropoietin, and granulocyte colony stimulat-ing factor (G-CSF) have been studied. Thesepharmaceuticals have been linked covalently tothe vitamin B12 molecule (Russell-Jones, 1998;Russell-Jones and Alpers, 1999). The system reliesupon the natural uptake mechanism for vitaminB12 to co-transport peptides and proteins linkedto vitamin from the intestine into the circulation.To maximize the potential of the delivery system,the pharmaceutical is being incorporated withinbiodegradable nanoparticles, and coated with vi-tamin B12 (Russell-Jones et al., 1999). This hasadvantages of protecting the pharmaceutical fromproteolysis within the intestine, of amplifying theuptake capacity of the oral delivery system, andof eliminating the need for conjugation of phar-maceuticals to vitamin B12. There have also beenother proposals utilizing the pre-existing mecha-nisms of molecule delivery within the body, suchas drug targeting biodegradable nanoparticlescoupled to folic acid (Stella et al., 2000).

3.1.4. Other applicationsIn addition to oral administration (Langer,

2000), the use of nanoparticles for nasal (Illum etal., 2001) and ophthalmic delivery of drugs(Bourlais et al., 1998; de Campos et al., 2001) hasbeen investigated. Nanoparticles have enabledcrossing the blood–brain barrier that representsan insurmountable obstacle for a large number ofdrugs, including antibiotics, antineoplastic agents,and a variety of central nervous system activedrugs, especially neuropeptides (Kreuter, 2001;Schroeder et al., 1998). Furthermore, nanosizecarriers of such molecules as vitamins, vitamin A


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

4. Towards the future

Medical diagnosis with proper and efficient de-livery of pharmaceuticals are the medical fieldswhere nanosize materials have found practicalimplementations. However, there are several otherintriguing proposals for practical applications ofnanomechanical tools into the fields of medicalresearch and clinical practice. Such nanotools stillawait construction, and at present they are rathermore like a fantasy. Nevertheless, they might bevery helpful, and become a reality in the nearfuture.

One function of nanodevices in medical sciencescould be the replacement of defective or incor-rectly functioning cells, such as the respirocyteproposed by Freitas (1996a, 1998). This artificialred blood cell is theoretically able to provideoxygen and can do it even more effectively thanan erythrocyte. It could replace defective naturalred cells in blood circulation. An onboardnanocomputer and numerous chemical and pres-sure sensors enable complex device behaviors thatare remotely reprogrammable by the physician viaexternally applied acoustic signals. Primary appli-cations of respirocytes may include transfusableblood substitution, partial treatment for anemia,prenatal/neonatal problems, and lung disorders.This artificial respirocyte could support oxygendistribution, improving the levels of available oxy-gen despite reduced blood flow. Thus the nextapplication phase of nanomachines could beproviding metabolic support in the event of im-paired circulation.

It has also been postulated that nanomachinescould distribute drugs within the patient’s body.Such nanoconstructions could deliver medicinesto particular sites, making more adequate andprecise treatment possible (Fahy, 1993a,b; Triggle,1999). Such devices would have a small computer,several binding sites to determine the concentra-tion of specific molecules, and a supply of some‘poison’ that could be released selectively. Similar

machines equipped with specific ‘weapons’ couldbe used to remove obstructions in the circulatorysystem or identify and kill cancer cells.

The other important application of nanotech-nology relates to medical research and diagnos-tics. Nanorobots, operating in the human body,could monitor levels of different compounds andstore that information in internal memory. Theycould be used to rapidly examine a given tissuelocation, surveying its biochemistry, biomechan-ics, and histometric characteristics in greater de-tail. This would help in better disease diagnosing(Freitas, 1996a; Lampton, 1995). The use of nano-devices would give the additional benefits of re-duced intrusiveness, increased patient comfort,and greater fidelity of results, since the targettissue can be examined in its active state in theactual host environment.

The use of nanoscale machines cruising throughour bodies, attacking viruses and diseases, killingcancer cells, and repairing damaged cells and tis-sues, may seem now to be science fiction. How-ever, in researchers’ imaginations such machineshave already appeared and now there are ‘only’ afew steps to make these designs come true.

Acknowledgements

The authors are grateful to Dr J. Johnson andDr T. Kubik for their help in the final prepara-tions of the manuscript.

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