Review

General

Nanotechnology and medicine

Dwaine F Emerich† & Christopher G Thanos
†Sertoli Technologies, Inc., 245 Armington Street, Cranston, RI 02905, USA

1. Introduction Nanotechnology, or systems/device manufacture at the molecular level, is a

2. Manufacturing at the
multidisciplinary scientific field undergoing explosive development. The gen-
molecular level esis of nanotechnology can be traced to the promise of revolutionary
advances across medicine, communications, genomics and robotics. On the
3. Nanoparticles for diagnostic
surface, miniaturisation provides cost effective and more rapidly functioning
and screening purposes
mechanical, chemical and biological components. Less obvious though is the
4. Development of artificial
fact that nanometre sized objects also possess remarkable self-ordering and
receptors
assembly behaviours under the control of forces quite different from macro
5. DNA sequencing using objects. These unique behaviours are what make nanotechnology possible,
nanopores and by increasing our understanding of these processes, new approaches to
6. Drug delivery enhancing the quality of human life will surely be developed. A complete list
7. Gene therapy of the potential applications of nanotechnology is too vast and diverse to dis-
cuss in detail, but without doubt one of the greatest values of nanotechnol-
8. Next generation drug
delivery devices
ogy will be in the development of new and effective medical treatments (i.e.,
nanomedicine). This review focuses on the potential of nanotechnology in
9. Tissue engineering
medicine, including the development of nanoparticles for diagnostic and
10. Expert opinion screening purposes, artificial receptors, DNA sequencing using nanopores,
manufacture of unique drug delivery systems, gene therapy applications and
the enablement of tissue engineering.

Keywords: drug delivery, encapsulation, gene therapy, nanotechnology, tissue engineering

Expert Opin. Biol. Ther. (2003) 3(4):655-663

1. Introduction

In 1959, at the annual meeting of the American Physical Society, the Nobel Laureate
Richard Feynman gave a lecture entitled ‘There’s plenty of room at the bottom,’ out-
lining the principle of manipulating individual atoms using larger machines to manu-
facture increasingly smaller machines [1]. This concept was echoed by the more recent
Congressional testimony of Richard E Smalley [2], winner of the 1996 Nobel Prize in
Chemistry, in which he stated ‘We are about to be able to build things that work on
the smallest possible length scales, atom by atom’. ‘There is a growing sense in the sci-
entific and technical community that we are about to enter a golden new era,’ he
stated. The scientific underpinnings of this new era are referred to as nanotechnology.
The term nanotechnology is derived from the Greek word nano, meaning dwarf,
and applies the principles of engineering and manufacturing at a molecular level.
The excitement generated by nanotechnology can be traced to the promise of revo-
For reprint orders, please lutionary changes in medicine, robotics and communications. This review focuses
contact: on what might be the greatest value of nanotechnology; the development of effective
[email protected] clinical treatments or nanomedicine. Nanomedicine depends on several overlapping
molecular technologies, which are themselves subsumed within young, but progres-
sively developing fields, including:
• the construction of nanoscale-sized structures for diagnostics, biosensors and local
drug delivery
Ashley Publications • the ongoing revolution in genomics, proteomics and nanoengineered microbes
www.ashley-pub.com
• the creation of molecular machines or medical nanorobots capable of identifying and
eliminating host pathogens, replacing/repairing cells or cellular components in vivo

2003 © Ashley Publications Ltd ISSN 1471-2598 655

Nanotechnology and medicine

This review highlights some of the recent advances in nan- these nanotweezers, 500-nanometre clusters of polystyrene
otechnology, with specific reference to those disciplines spheres have been grasped and moved. While this system cur-
most likely to benefit in the intermediate term, including rently uses multi-walled nanotubes to operate at the scale of
diagnostics and screening, drug delivery, gene therapy and cellular substructures, the future use of single-walled nano-
tissue engineering. tubes will likely produce nanotweezers small enough to grab
individual macromolecules.
2. Manufacturing at the molecular level
3.Nanoparticles for diagnostic and
Building anything to the scales required by nanotechnology screening purposes
presents enormous technical challenges. Manipulating matter
at the nanometre scale is important for many electronic, One of the first applications of nanotechnology will likely be
chemical and biological advances, but present solid-state fab- to improve the use of fluorescent markers for diagnostic and
rication methods do not reproducibly achieve dimensional screening purposes. While fluorescent markers are routinely
control at the nanometre scale. Currently, there are two gen- used in basic research and clinical diagnostic applications,
eral approaches to manufacturing at the nano level. The first there are several inherent disadvantages with current tech-
is referred to as self-assembly and takes advantage of some of niques, including the requirement for colour-matched lasers,
the natural properties of DNA. Strands of DNA are relatively the fading of fluorescence even after a single use and the lack
rigid polymers that tend to self assemble and interact with of discriminatory capacity of multiple dyes due to the ten-
other DNA strands in predictable ways [3]. DNA is also easily dency of the different dyes to bleed together. Fluorescent nan-
manipulated enzymatically and complimentary strands of oparticles potentially overcome these issues. One iteration of
DNA have been shown to assemble or ‘zip’ themselves up into this technology uses ‘Quantum dot’ nanocrystals which are
increasingly complex shapes including squares, three-dimen- extremely small (i.e., several nanometres in diameter) and
sional stick-figure cubes and truncated octahedrons consisting manufacturable in a nearly unlimited range of sharply defined
of hundreds to thousands of nucleotides. The self assembly colours [101]. The particles are excitable using white light and
properties of DNA have also been used to demonstrate that can be linked to biomolecules to form long-lived sensitive
(a) two rigid double-stranded DNA arms will rotate between probes to identify specific compounds. In principle, separate
fixed positions when a positively charged cobalt compound is biological events can be monitored by simultaneously tagging
introduced into the solution surrounding the molecules [4]; each biological component (e.g., different proteins or DNA
(b) three single strands of artificial DNA [5] will self-assemble sequences) with nanodots of a specific colour. Aside from the
to form a V-shaped structure capable of opening and closing clinical potential of these particles, their research value
with the addition or subtraction of additional DNA strands; includes the possibility of simultaneously tagging multiple
and (c) the enzyme ATPase will incorporate non-biological biomolecules both on and inside cells to monitor the complex
components, such as silicon nitride bars 100 nm in length, to cellular changes and events associated with disease, providing
form artificial molecular motors capable of rotating at up to valuable clues for the development of future pharmaceuticals
200 revolutions per minute [6,7]. Self-assembly processes are and therapeutics.
also applicable for the configuration of larger scale (millime- A related technology called Probes Encapsulated by Biologi-
tre) structures including three-dimensional shapes [8]. In some cally Localized Embedding (PEBBLES), pioneered by Raoul
cases, the final configuration is influenced by subtle changes Kopelman, allows dye-tagged nanoparticles to be inserted into
within the local chemical environment [4,8]. living cells to monitor metabolism or disease conditions [10,11].
The second general category of nanomanufacturing As one example, this system was recently used to quantify zinc
involves positional assembly. Building increasingly complex levels within living cells. The sensing components were
structures will be impossible using only self-assembly proc- entrapped within a polymer matrix using a microemulsion
esses to accurately and spontaneously position the individual polymerisation process that produced spherical sensors with a
components. Instead, positional assembly, or the means to size of 20 – 200 nm. The system was highly sensitive (capable
select and place parts into the appropriate area of the of detecting 4 – 50 µM Zn2+), had a rapid response time
designed system, will be needed. Probably the most recog- (< 4 seconds) and used a reversible and photostable nanosen-
nised example of this area of research is the development of sor that was insensitive to interference from intracellular or
nanotweezers by Philip Kim and Charles Lieber [9]. The tips extracellular proteins. A similar application uses superpara-
of the tweezers are formed by electrically controlled carbon magnetic nanoparticles as magnetic resonance (MR) contrast
nanotubes. A voltage is applied across the electrodes, causing agents for imaging specific molecular targets. Wunderbald-
one nanotube arm to develop a positive electrostatic charge inger et al. characterised the physical and biological properties
and the other to develop a negative charge. The attractive of MION-47 and amino-crosslinked iron oxide (-CLIO) nan-
force can be increased or decreased by varying the applied oparticles that serve as precursors for the synthesis of targeted
voltage. For instance, 8.5 volts completely closes the arms, MR contrast agents, and Tat-CLIO, a nanoparticle used to
while lower voltages give different degrees of grip. Using label cells [12]. Following intravenous injection, the plasma

656 Expert Opin. Biol. Ther. (2003) 3(4)

 Emerich & Thanos

half-lifes of MION-47 and amino-CLIO were > 10 h. The bolus IV dose to monitor myocardial perfusion and ventricu-
attachment of tat peptides to amino-CLIO significantly lar wall motion over the course of 10 min, and it may provide
reduced the circulating half-life to < 1 h and resulted in the a first detailed look at real-time haemodynamics in the
greatest accumulation of MION-47, amino-CLIO and Tat- human heart.
CLIO in the liver, spleen and lymph nodes. Furthermore, 24 h
after injection of amino-CLIO, the nanoparticles were con- 4. Development of artificial receptors
centrated in the endothelial and Kupffer cells surrounding
hepatic blood vessels, while Tat-CLIO was present throughout An early goal of nanomedicine is to provide insight into how
the parenchyma. Future approaches using peptides as compo- biological molecular receptors function and then to build arti-
nents of nanoparticles might make it possible to design sensors ficial binding sites capable of specifically recognising proteins.
to detect macromolecules present in specific intracellular com- For instance, Ratner and his colleagues [25] used a new radiof-
partments. Other applications include using gold nanoparti- requency-plasma glow-discharge process to imprint a polysac-
cles for DNA diagnostics [13] and DNA microarrays for charide-like film with nanometre-sized pits in the shape of
genotypic analysis for both diagnostic purposes and assess- protein molecules including albumin, immunoglobulin,
ment of drug responses [14]. fibrinogen, lysozyme, ribonuclease A, alpha-lactalbumin and
A final application of fluorescent nanoscale devices worth glutamine synthetase. Electron spectroscopy for chemical
mentioning might allow transdermal monitoring of changes analysis and time-of-flight secondary ion mass spectrometry
in interstitial fluid constituents. Electrostatic, self-assembly confirmed the saccharide covering of imprint surfaces and the
processed, solid nanoparticles, coated with fluorescent removal of template proteins. 125I-labelled protein adsorption
enzyme-containing thin films and hollow micro/nanocap- from single solutions showed a similar amount of protein was
sules containing fluorescent indicators and enzymes or glu- adsorbed to its own imprint as to the imprint of another pro-
cose-binding proteins, are being used to monitor glucose tein. However, more protein remained on the former surface
concentrations [15]. Nanoengineering of the coated colloids than on the latter following elution with the detergents
and microcapsules allows precision control over optical, Tween 20 or sodium dodecyl sulfate. Competitive adsorption
mechanical and catalytic properties to achieve sensitive of a binary protein mixture showed a highly preferential
response using a combination of polymers, fluorescent indi- adsorption of template protein to the corresponding imprint.
cators and glucose-specific proteins. Challenges to in vivo use Engineered surfaces like these may one day be used for rapid
include understanding of material toxicity and failure modes biochemical separations and assays, and in biosensors and
and determining methods to overcome fouling, protein inac- chemical sensors, because of their ability to selectively adsorb
tivation and material degradation. specific proteins from solutions whose complementary shape
One very important implementation of nanoscale technol- has been imprinted on that surface.
ogy for clinical evaluation comes in the form of perfluorocar-
bon nanoparticles for molecular imaging. Several groups have 5. DNA sequencing using nanopores
demonstrated that polymeric nanospheres are capable of selec-
tively targeting different tissues for the purpose of imaging, The flow of DNA through nanopores can be externally regu-
which also has enormous implications in targeted therapy. lated to discriminate low copy numbers of DNA polymers,
Work by Lanza et al. [16-18] has demonstrated the evolution of permitting very rapid genome sequencing. Initial attempts
one of these nanoparticle systems from the initial inception as used a molecular nanosieve consisting of an array of cylindrical
a contrast agent to the more advanced application of targeted gold nanotubules with inner diameters as small as 1.6 nm [26].
local drug delivery. Through MR imaging, this group has When the tubules were positively charged, positive ions were
shown localisation of ligand-linked perfluorocarbon excluded and negative ions were transported through the
nanoemulsions (250 nm diameter) to various sites including membrane. In contrast, when the membrane was negatively
thrombi, and neovascularisation during tumourigenesis both charged only positive ions passed through. Electric fields have
in vitro and in vivo [19-24]. By directing the delivery vehicle to a also been used to push RNA and DNA polymers through the
site-specific target, the formulation is capable of delivering central nanopore of an alpha-haemolysin protein channel
drug locally without the need for internalisation, a process mounted in a lipid bilayer [27]. The individual nucleotides
termed contact-facilitated drug delivery. In studies to date, the comprising the polynucleotide strands pass single-file through
group has been able to confirm local targeted delivery through the 2.6 nm-wide nanopore and changes in ionic current are
imaging as well as provide quantitative evidence by resolving used to measure the length of the polymer. Single-channel
individual nanospheres in real-time. recording of the translocation duration and current flow dur-
Another group has recently brought a similar product ing traversal of individual polynucleotides yields a unique pat-
through Phase III clinical trials in the form of biodegradable tern of events for each of the several polymers tested. Statistical
microspheres. Walovitch et al. [102] used poly(lactide-co-gly- data derived from this pattern of events further demonstrates
colide) (PLGA) particles encapsulating a low solubility gas as that nanopores can be used to distinguish between polynucle-
a contrast agent for echocardiography. This technique uses a otides of similar length and composition that differ only in

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Nanotechnology and medicine

sequence. This technology has been refined to discriminate bioavailability. Microsphere formulations provide protection
between pyrimidine and purine nucleotide bases along a single for agents susceptible to degradation or denaturation in
RNA molecule and to distinguish between DNA chains of regions of harsh pH, and also prolong the duration of expo-
similar length and composition which differ only in base pair sure of a drug by increasing retention of the formulation
sequence [27]. DNA-nanopores are able to discriminate through bioadhesion [30,31]. Particle formulations smaller
between individual DNA strands up to 30 nucleotides in than one micron have a higher associated degree of difficulty
length, differing by a single base substitution, as shown by the for fabrication as well as administration (e.g., aggregation).
detection of a drug resistance-conferring mutation in the However, one principle advantage of nanoparticles over
reverse transcriptase gene of HIV [28]. This technique was also microparticles is the ability to cross membrane barriers, par-
used to sequence a complete codon in an individual DNA ticularly in the absorptive epithelium of the small
strand tethered to a nanopore. In principle, nanopore detec- intestine [32]. Several groups have used both non-resorbable
tion and characterisation of single molecules represents a new and biodegradable nanospheres to deliver small molecules,
method for directly reading information encoded in linear proteins and other therapeutics. The attraction of biode-
polymers. If single-nucleotide resolution can be achieved, it is gradable nanospheres centres on the principle of enhanced
possible that nucleic acid sequences could be determined at bioavailability through uptake, followed by degradation and
rates exceeding 1000 bases per second [29]. Because nanopores disappearance of the vehicle from the system.
can rapidly discriminate and characterise unlabelled DNA Another broad application of nanosphere uptake is the
molecules at low copy number, refinements of these experi- delivery of antigens for vaccination. Mucosal immunity is
mental approaches could eventually provide a low-cost high- extremely important in disease prevention, but continues to be
throughput method of analysing DNA polynucleotides. limited by both degradation of the vaccine and limited uptake.
Future nanodevices may combine voltage gating with pore Recent advances in encapsulation and development of suitable
size, shape and charge constraints to achieve precise control of animal models have demonstrated that micro and nanospheres
ion transport with significant molecular specificity. are capable of enhancing immunisation. It has been shown
that M cells in the Peyer’s Patches of the distal small intestine
6. Drug delivery are capable of engulfing large microspheres [33-35] and recent
studies have explored the benefits of nanoencapsulation.
In the last decade, great advances have been made in the field Lutsiak et al. [36] have recently demonstrated uptake of PLGA
of nanotechnology and nanofabrication that have had an nanospheres by human dendritic cells in vitro. PLGA nano-
immediate impact on the field of drug delivery. Techniques spheres loaded with tetramethylrhodamine-labelled dextran
have shifted from microfabrication and micromachining (e.g., were prepared using a solvent evaporation technique and
the osmotic pump) to designs ranging from secondary con- administered to cultures of dendritic cells and macrophages
structs on the nanometre level (e.g., microspheres) to delivery established from peripheral blood leukocytes. After 24 h, con-
systems composed entirely of submicron components. The focal microscopy revealed the internalisation of these nano-
development of delivery systems for small molecules, proteins spheres by dendritic cells as well as macrophages, with the
and DNA has been impacted to an enormous degree by nan- same level of uptake in each cell type. This work has
otechnology and has led to the development of entirely new implications in selective activation of a T cell-mediated
and previously unpredicted fields. Formulation science has immune response.
formed a unique bridge with computer technology, leading to Nanoparticles have also been used for pulmonary delivery.
the creation of a controlled-release microchip capable of infi- Bustami et al. showed that proteins in aqueous solutions can
nite modulation. Tissue engineering applications have also be precipitated into 100 – 500 nm particles for aerosolised
tended towards the development and implementation of nan- delivery without significant loss of biochemical integrity [37].
ometre-sized components. Barriers are being overcome that This work used a novel fabrication process called the Aerosol
provide a better understanding of normal physiological proc- Solvent Extraction System to generate aerosolised nanoparti-
esses by creating artificial cells with appropriate physiologic cles from lysozyme, albumin, insulin and recombinant human
properties. Also, in the age of genetic manipulation and gene deoxyribonuclease, and tested the biological protein integrity
delivery, transfection systems on nanoscales are being custom- using size exclusion chromatography. Recombinant human
tailored using polymers for different applications. These deoxyribonuclease was the only protein denatured by the
advances are only several out of many that have been recently process, and all aerosolised particles showed very low levels of
accomplished. The scope of this review does not permit a aggregation. In similar work, Loscertales et al. produced mon-
detailed discussion of every advance in the field, but rather odisperse aerosols in the range of 150 nm using a novel encap-
touches on several key areas of this rapidly growing segment sulation technique based on electrified coaxial liquid jets [38].
of nanotechnology. This technique allows for the precise control of the thickness
Nanotechnology is opening new therapeutic opportuni- of either the inner or outer microcapsule layer, as well as the
ties for a large number of agents that cannot be used effec- overall size of the emulsion droplet, by simply adjusting the
tively as conventional oral formulations, due to poor concentration, flow rate or voltage used in the process.

658 Expert Opin. Biol. Ther. (2003) 3(4)

 Emerich & Thanos

7. Gene therapy negatively charged phosphate groups on the DNA backbone

and the positively charged amino groups on the polymer.
Gene therapy is dependent on the entry of the vector into the The complex is formed by simply mixing the components in
cell while protecting the contents from the harsh environment an aqueous solution. Polyamidoamines (PAMAM) are the
of the cytoplasm. Next, transport of the gene into the nucleus most often used and characterised dendrimers for gene deliv-
must occur without imparting damage from the vehicle itself. ery and several groups have recently demonstrated their effi-
One of the most investigated approaches to gene therapy uses cacy. Kukowska-Latallo et al. [44] used ninth-generation
liposomes as submicron delivery vehicles consisting of a lipid PAMAM dendrimers complexed to the pCF1CAT plasmid
shell surrounding a core containing a therapeutic molecule or for intravascular and endobronchial delivery of chlroam-
gene. Liposomes are particularly useful as gene therapy phenicol acetyl-transferase (CAT) to eventually treat cystic
devices due to their ability to pass through lipid bilayers and fibrosis. Intravenous administration of the complex showed
cell membranes, and several groups have recently reported transgene expression in the lung with peaks at 12 – 24 h and
convincing results following local delivery. Liu et al. have 3 – 5 days. The 114 Å dendrimer–plasmid complex was
shown that composite liposomes containing poly(cationic completely localised to the lung, whereas the naked plasmid
lipid) and cholesterol showed much higher transfection in the was randomly distributed. In comparison, endobronchial
liver than naked DNA alone [39]. This work focused on lipo- delivery of naked plasmid was more effective than the den-
plexes consisting of poly(cationic lipid), cholesterol and DNA drimer complex. Maksimenko et al. [45] have also shown
injected directly into portal circulation following a partial improved transfection using PAMAM conjugates. This study
hepatectomy. The reporter gene expression (luciferase) was showed that the transfection of several cell lines by plasmid
observed to be much higher in these lipoplexes than in naked cytomegalovirus β-galactosidase plasmid–dendrimer com-
DNA alone. Targeted therapy can also be achieved using lipo- plexes was enhanced by the presence of anionic oligomers
somes. Zhang et al. showed that pegylated liposomes linked to including oligonucleotides or dextran sulfate. The degree of
a monoclonal antibody for the human insulin receptor led to enhancement was dependent on the size and charge of the
widespread reporter expression in the brain of rhesus additive, with 35 – 50 phosphate group oligonucleotides
monkeys [40]. Plasmids encoding luciferase or β-galactosidase providing the highest efficiency.
were administered via the ‘immunoliposome’ through the
common circulation and were localised primarily to the brain. 8. Next generation drug delivery devices
Such complexes give a hint at the future of targeted therapy
and the importance of nanometre-sized constructs for the Just as the potential of micro and eventually nanospheres has
advancement of molecular medicine. become apparent in the last decade, we are now beginning to
Gene delivery using a polymeric vehicle has also been witness the emergence of novel technologies that offer an
explored recently. Polymeric nanospheres are capable of translo- entirely new approach to drug delivery. Such technologies are
cation into the cytoplasm of a cell, but transport to the nucleus a result of the trend towards multi-disciplinary approaches to
has not been established. Cohen-Sacks et al. [41] have recently pharmaceutics and, although there are certainly many poten-
carried out experiments to characterise the delivery of 300 nm- tial therapies in development, two are of particular relevance.
diameter nanospheres loaded with platelet-derived growth fac- As a pioneer of drug delivery therapies and devices,
tor beta-receptor antisense for the treatment of restenosis. This Dr R Langer and his colleagues have consistently provided
group observed cell internalisation as well as inhibition of the field with a wealth of innovation and palpable technolo-
smooth muscle cell proliferation and an ∼ 22% reduction in gies. One recent approach by this group has focused on the
restenosis compared to the control. Berton et al. [42] have also integration of controlled-release drug reservoirs with a
demonstrated successful delivery of oligonucleotides. This microchip [46], with operational elements of both disci-
group characterised the ability of PLGA nanospheres to plines. The chip is composed of a silicon wafer containing
deliver phosphorothioate oligonucleotides with subsequent up to 34 reservoirs, each occupying a volume of 25 nl,
inhibition of viral HIV-1 production. Plasmid DNA can also sealed with a gold membrane anode 300 nm in thickness.
be delivered by biodegradable nanospheres. Perez et al. [43] Drug is released from a reservoir when a current is applied
constructed poly(lactic acid)/poly(ethylene glycol) composite between the gold membrane and an embedded cathode.
nanospheres with mean diameter < 300 nm and showed that This device has unlimited potential for modulation of
modulation of release could be achieved with the addition of release, with the ability to combine modalities within each
either poly(vinyl alcohol) or poly(vinylpyrrolidone) or both. reservoir as well as the control of each individual pulse of
Dendritic polymers provide another avenue for delivery of drug release. Although only in its early stages, the chip has
genes. They can form extremely small particles, in the order of shown extremely promising results with fluorescent tracers
Angstroms, and have been shown to be effective as DNA-con- and is in continued development.
jugates. The resulting dendrimer–DNA complex differs from Nanotube technology is another vast field of rapid develop-
encapsulation in that the primary interaction causing gene ment and discovery that provides attractive alternatives to
retention is caused by electrostatic interactions between the standard formulation techniques. Nanotubes have large relative

Expert Opin. Biol. Ther. (2003) 3(4) 659

Nanotechnology and medicine

internal volumes, can be functionalised either on the internal and the murine NR6 fibroblast cell line was cultured onto the
or external surface and can be made using a variety of tech- surface. The density of RGD was significantly reduced for cell
niques [47]. One potential advantage of the cylindrical geome- migration to occur in this arrangement. Osteogenic cells have
try of nanotubes is the ability to introduce a therapeutic agent been cultured in a three-dimensional nano-hydroxyapatite/col-
following fabrication of the vehicle, whereas most encapsula- lagen matrix by Du et al. [55]. This matrix is precipitated such
tion methods require co-precipitation into a homogeneous that hydroxyapatite crystals are uniformly distributed in a
matrix. Whether this is an advantage remains to be seen. One matrix of collagen, seemingly ideal for bone construction.
of the fabrication techniques includes self-assembling lipid Bone-derived mesenchymal cells reached confluence at
microtubes, which have been shown to effectively release testo- 3 weeks, with a tissue morphology consisting of a mass of
sterone in rats [48]. Goldstein et al. demonstrated that testoster- polygonal cells over which spindle-shaped cells migrated, simi-
one could be covalently bound with an ester linkage to a lar to the three-dimensional construction of bone. Nanofibril-
glutamide core lipid and the resulting complex could form lar extracellular matrices have also been created using a novel
nanotubes. The in vivo release profiles were biphasic with an technique employing porogenic materials and polymers [56].
initial burst followed by a more sustained release. Another Sugar particles were melted and extruded into nanofibres
method of fabrication used for drug delivery involves synthe- which were subsequently suspended in an organic solution of
sising carbon nanotubes using fullerene. These nanotubes poly(lactic acid) in tetrahydrofuran. The solution was dripped
range from one to tens of nanometres in diameter, and their into the dissolved porogen and gelation was induced with
length can range from several to hundreds of microns. Recent freezing. After leaching and lyophilisation, the resulting
studies have shown the ability to functionalise the external sur- porous matrices were extremely homogeneous and contained
face for subsequent covalent attachment of a drug [49]. a true three-dimensional interconnecting nanoporous matrix.
Inverse patterning has also been explored in recent studies
9. Tissue engineering by Folch et al. [57]. In this method, a polymeric membrane is
applied to a substrate and cells are allowed to spread across the
Designing a biological system capable of reproducing a physi- surface. After a confluent layer has been achieved, the mem-
ologic event requires not only that the system give the desired brane is removed and the resulting cell pattern is left on the
outcome, but also that the outcome is reached in a suitable surface. Patterned cells were created on polystyrene, glass and
and repeatable fashion. Historically, this is one of the great collagen, with either flat or curved geometry. This technique
challenges of tissue engineering, but a new level of control is has the potential to facilitate the development of a variety of
being gained with advancements in biohybrid processing cell constructs as well as the creation of biohybrid organs
techniques and nanoscale fabrication and modification. Scaf- using multidimensional applications.
folds capable of sustaining dense populations of cells for The techniques described above are extremely relevant for
extended periods are being developed, new materials are being the development of artificial tissues and organs. Still, new
used in a variety of matrices and new constructs are being advances are leading to models for individual cells and Cans
engineered that will lead to the eventual development of the et al. [58] have cleverly characterised the process of exocytosis
artificial cell/organ. using liposomes and nanotubes. In an effort to understand
The response of cell motility and metabolism to changes in and reproduce neuronal transmission, this group has effec-
substrates has been thoroughly studied in the past decade. tively simulated natural physiology using a system composed
Size, structure, geometry, integrin-binding and other factors of liposome–nanotube networks controlled by electroinjec-
have all been investigated. Various techniques have been tion. A small vesicle is introduced into a liposome that is con-
employed to create micropatterned surfaces of different mate- nected to a nanotube, which together resembles a
rials to study cell behaviour, including microlithography [50], neurotransmitter vesicle and elongated fusion pore and forms
microcontact printing [51], hydrolytic etching [52] and many the liposome–nanotube network. Measured with fluorescence
others. Thompson et al. [50] demonstrated that in the presence microscopy and amperometry, this model was compared to
of patterned stripes of bovine serum albumin and laminin, dopamine release from PC12 cells and was found to be slower,
Schwann cells aggregate preferentially on the laminin regions. but qualitatively similar in magnitude. The system can be fur-
Lee et al. [51] showed that using microcontact printing to cre- ther controlled to investigate individual phases of this process.
ate shapes composed of inhibitory molecules affects the mor-
phology and growth of retinal pigmented epithelium. Such 10. Expert opinion
forms of surface modification have evolved into even smaller-
scale attempts to control cell culture. The multidisciplinary field of nanotechnology is making the
Irvine et al. [53,54] have developed a novel system of science of the almost incomprehensibly small device closer
poly(methyl methacrylate-r-polyoxyethylene methacrylate) and closer to reality. The effects of these developments will, at
comb polymers that present Arg-Gly-Asp (RGD) peptides in some point, be so vast that they will likely affect virtually all
nanoclusters on the surface. The RGD adhesion ligand was clus- fields of science and technology. As such, nanotechnology
tered with a spatial distribution of 50 nm at varying densities holds the promise of delivering the greatest technological

660 Expert Opin. Biol. Ther. (2003) 3(4)

 Emerich & Thanos

breakthroughs in history. Here, several developments in the site of dysfunction. However, it is at this point that the indi-
field of nanotechnology are presented, with particular empha- vidual segments of nanotechnology for applications such as
sis on those likely to make rapid medical advances. Impor- gene therapy, tissue engineering and device manufacturing
tantly though, the scope of the nanotechnology does not require significant improvements. These include:
allow every promising development to be mentioned and sev-
• The identification of materials, whether raw or structured,
eral have not been addressed, including new liposomal-based
suitable for nanostructuring purposes. Nanotubes, nano-
technologies [59,60], MR imaging techniques for tracking stem
particles and fullerenes are becoming more established and
cell migration in vivo [61] and the use of microbubbles for con-
reproducible to manufacture and may be ideal for cell scaf-
trast imaging, drug and gene delivery [62-64]. The reader is,
folding, drug delivery and artificial tissues.
therefore, referred to these fascinating new technologies.
• Improved ability to manufacture and build complex shaped
Feynman aptly, if not presciently, described the use of
systems. Current self-assembly and positional-assembly
nanomachines with the characteristics of cells including the
approaches will not be adequate for many medical applica-
ability to move, synthesise and deliver substances and store
tions requiring a more sophisticated integration of biologi-
information. His lecture was even more outstanding given
cal and mechanical components.
that it preceded Moore’s law of microminiaturisation. How-
• Greater understanding of the normal developmental regu-
ever, Feynman never really elaborated on his idea and it wasn’t
lation of tissue and organ systems at the level of the individ-
until recently that nanotechnological development began to
ual cell.
move from blue sky to practicality.
• Insight into the process of moving individual atoms while
There is a double-edged sword that comes from the extent
maintaining their inherent physical nature.
of Feynman’s vision. His articulation of the concept of nan-
• Development of smart devices or systems for robotic uses,
otechnology immediately leads to predictions of miniature,
manufacturing materials that respond to external stimuli,
self-replicating robots capable of surveying and repairing the
molecular machines capable of working at the level of indi-
human body on a molecular level. Diseases from the common
vidual genes or atoms and self-replicating devices.
flu to cancer to coronary dysfunction are no longer health
issues. Whether this comes to pass or not we cannot predict, Some of the technologies for these areas are available or will
but it is interesting that typically conservative institutions be very soon; others are in development or are still conceptual
such as XEROX, IBM, the National Aeronautics and Space and their usefulness is unknown. Many of the difficulties we
Administration’s Ames Research Center and the National Sci- will face are a function of our lack of understanding of the
ence Foundation are actively involved in nanotechnology three-dimensional physical properties of atom–atom relation-
research. What does seem fairly certain, though, is there are ships and normal, not to mention abnormal, biological proc-
medical benefits that will occur in the relatively short-term, esses. Many of the developments in molecular manufacturing
some of which have been focused on here. The initial impact arise because of accident and not because of careful planning.
of nanotechnology will likely be felt in diagnosis and disease Even the development of the so-called buckminsterfullerene
screening and continued elucidation of normal and patholog- was largely accidental.
ical cellular function. Over the last 20 years, many of the In the end though, the scope of nanotechnology makes it
major breakthroughs in medicine have come not from treat- uniquely suited to provide contemporary and profound med-
ment but from imaging, identification and characterisation of ical applications, while also providing endless opportunities
disease processes. The ability to continue these developments to set the scientific bar high, ignore the experts and in all
at the level of individual cells will dovetail nicely with efforts likelihood come up with inventions that will be among the
to deliver potentially efficacious therapeutics directly to the greatest in history.

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