REVIEW

Micro- and nanofabrication methods

in nanotechnological medical and pharmaceutical
devices

Tania Betancourt Abstract: Micro- and nanofabrication techniques have revolutionized the pharmaceutical
Lisa Brannon-Peppas and medical fields as they offer the possibility for highly reproducible mass-fabrication of
systems with complex geometries and functionalities, including novel drug delivery systems
Department of Biomedical
Engineering, University of Texas and bionsensors. The principal micro- and nanofabrication techniques are described, including
at Austin, TX, USA photolithography, soft lithography, film deposition, etching, bonding, molecular self assembly,
electrically induced nanopatterning, rapid prototyping, and electron, X-ray, colloidal monolayer,
and focused ion beam lithography. Application of these techniques for the fabrication of
drug delivery and biosensing systems including injectable, implantable, transdermal, and
mucoadhesive devices is described.
Keywords: microfabrication, nanofabrication, medical devices, drug delivery, biosensors

Introduction
Micro- and nanodevices have many advantages over their macroscale counterparts.
For instance, miniaturization allows for the manufacture of portable, hand-held,
implantable, or even injectable devices. In addition, as a result of their minute size,
these devices need less sample or reagent for analysis or operation, saving money
and time. Moreover, where materials and/or processes are inhibited by lengthy diffu-
sion times, miniaturization provides a mechanism for abbreviating these. A notable
example where these microdevices allow for significant advantages over traditional
technologies is in medical care. For example, point-of-care diagnostic testing, which
is testing performed at the patient’s bedside, permits physicians to diagnose a patient’s
conditions more rapidly than conventional lab-based testing. By using these devices to
reduce the time to diagnoses, the physician is able to make better patient management
decisions leading to improved patient outcomes and reduce the overall cost of care.
Advances in microelectronics and biosensor tools have been instrumental in facilitating
the development of these point-of-care diagnostic devices.
Microfabrication techniques were developed for applications in the semiconductor
industry and are, consequently, not specific for biological or medical applications.
Nonetheless, both micro- and nanofabrication have offered a number of possibilities for
the study of chemical, biological, and physical processes at the cellular and molecular
scale, and for the design of synthetic devices capable of interacting with biological
systems at these levels.
Correspondence: Lisa Brannon-Peppas Some of the advantages of micro- and nanofabricated devices include the ability to
Department of Biomedical Engineering, control the features to the nanometer scale for reproducible mass production of struc-
The University of Texas at Austin,
1 University Station C0300, Austin,TX tures and devices, the ability to miniaturize already-existing systems for the study of
78712, USA cellular or molecular processes, the capacity of including electronics within structural
Tel +1 512 471 4348
Fax +1 512 471 4348
devices through the use of the well-developed semiconductor techniques, and the high
Email [email protected] throughput possible with some of the micro- and nanofabrication methods.

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© 2006 Dove Medical Press Limited. All rights reserved
Betancourt and Brannon-Peppas

The integration of the knowledge gained from micro- and The microfabrication process utilizes these techniques in a
nano-fabrication can lead to design principles for nanode- sequential manner to produce the desired structure. These
vices that can detect substances, analyze their environment, structures can be built within the bulk of a substrate material
and perform tasks such as the release of a specific molecule. in what is known as bulk micromachining, or on the surface
These vehicles will combine responsive polymers, nanopar- of the substrate through surface micromachining (Voldman
ticles, nucleotides, and micro-electromechanical systems et al 1999). In most cases, however, a combination of bulk
(MEMS) elements. and surface micromachining is utilized in the fabrication of
Expertise in combining MEMS systems with environ- the desired system.
mentally sensitive polymers has led to the design of The most important microfabrication techniques are
controlled release systems. For example, research in physi- photolithography, soft lithography, film deposition, etching,
ologically responsive materials shows how it is possible and bonding. Photolithography is used to transfer a user-
to design devices which are responsive to changes in the generated shape onto a material through the selective
surrounding environment. Biological molecular recogni- exposure of a light sensitive polymer. Soft lithography
tion systems have been used widely in designing novel encompasses three different techniques which are all based on
devices such as DNA-fueled molecular machines (Seeman the generation and utilization of the mold of a microstructure
2003; Yurke et al 2003), tailored colloidal aggregates, out of poly(dimethylsiloxane). Film deposition, as its name
and biomolecular nanomechanical sensors. We have also suggest, consists of the formation of micron-thick films on the
expertise in the controlled release of therapeutics, and in surface of a substrate. Etching selectively removes materials
modifying the targeting and release properties of biodegrad- from the surface of the microdevice by either chemical or
able nanoparticles (Blanchette et al 2004; Brannon-Peppas physical processes. Finally, bonding adheres substrates
and Blanchette 2004). It will be important to integrate together with or without the use of intermediary layers. The
MEMS technology into the biological environment, as with following section will discuss these and other techniques in
polymeric nanoparticle delivery systems, and microfabri- more detail.
cated nano- and microcontainers with responsive delivery
systems (sensor-controlled delivery). Photolithography
Finally, investigation of intelligent nanoscale systems Photolithography is one of the most readily employed
with the ability of the molecules themselves to make deci- microfabrication techniques and is used to create patterns
sions is needed in our field. Nucleic acids are likely the best into a material. The photolithographic technique has been
candidates for sensing, transducing, deciding, and treating, as reviewed thoroughly previously (Voldman et al 1999; Li et al
demonstrated by nucleic acid “gates” that can sense analytes, 2003). The photolithographic process consists of a number of
integrate information, and carry out reactions that would in steps in which a desired pattern is generated on the surface of
turn lead to the release of therapeutic molecules, such as a substrate through exposure of regions of a light-sensitive
siRNAs. By integrating these technologies with novel drug material to ultraviolet (UV) light. Figure 1 summarizes the
delivery methods, it should prove possible to make extremely main steps followed in photolithography.
“smart” therapeutics. In the first step, a substrate material, such as silicone or
This review focuses on the diverse micro- and nano- glass, is coated with a layer of a photoresist, or light-sensitive
fabrication techniques available, and on the applications polymer. A photomask, made by patterning with an opaque
of these techniques into the construction of devices for material the desired shape on a glass dish or other transparent
medical applications. However, because of the vast number material, is placed on top of the substrate and photoresist. This
of techniques that have been developed in the recent years assembly is then irradiated with UV light, thus exposing the
for very specific applications, this review is by no means sections of the photoresist not covered by the opaque regions
exhaustive. of the photomask. Depending on the type of photoresist
utilized, the photoresist polymer will undergo one of two
Microfabrication techniques possible transformations upon exposure to light. When light
A number of techniques are used for the fabrication of illuminates a positive photoresist, the exposed regions break
micron-scale devices. Some of these techniques have been down and become more soluble in a developing solution. As
adopted from the well-established field of semiconductors, but a result, the exposed photoresist can be removed when in
others have been specifically developed for microfabrication. contact with the developing solution. A negative photoresist,

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Opaque Transparent UV Light variety of pattern attributes that are possible to obtain, both
Region Region
of which depend on the characteristics of the photomask.
Mask Nonetheless, this technique has the limitation of requiring
clean room processing.
Photoresist
Soft lithography
Soft lithography, similarly to photolithography, is a method
Substrate
also used to transfer a pattern onto a surface. It utilizes a
microstructure replica produced by molding a polymer, such
as poly(dimethyl siloxane) (PDMS) to a master, which is
Negative Positive
manufactured through other microfabrication techniques such
Photoresist Photoresist as photolitography (Li et al 2003). PDMS has been readily
used in the biomedical and pharmaceutical fields because
of its biocompatibility, and good thermal, mechanical and
Figure 1 Process of photolithography. A mask with opaque regions in the desired
optical properties. The main advantage of soft lithography is
pattern is used to selectively illuminate a light-sensitive photoresist. Depending on that once the reusable mold is made, none of the other steps
the type of photoresist utilized, it will become more soluble (positive photoresist)
or crosslinked (negative photoresist) after UV light exposure, thus generating the require clean room manipulation. As a result, it is a less
appropriate pattern upon developing. expensive technique that provides great resolution through
a simpler process (Li et al 2003). A great review on soft
lithographic methods was previously published (Xia and
on the other hand, becomes crosslinked upon exposure to Whitesides 1998).
light, thus becoming insoluble in the developing solution. There are three main soft lithography processes: micro-
Consequently, upon contact with the developing solution, stamping, stencil patterning, and microfluidic patterning.
only the parts not exposed to light will be removed. Figure 2 depicts these three microfabrication methods.
The resulting photoresist patterns are then used to protect
the covered substrate from etching, or from the deposition of
Photoresist
compounds or biomolecules on its surface. After the desired pattern

process is completed, the photoresist can be removed, leaving Plate to prevent

master coverage
the pattern design on the substrate. The technique used for Add
PDMS PDMS
photoresist removal usually consists of sonication in an PDMS

organic solvent, and may consequently be undesirable for

a number of systems, specially those containing biological
molecules. As an alternative, water-soluble photoresists have
been developed; however, concerns about the efficiency of
STENCIL
MICROFLUIDIC
water-soluble photoresist removal have been reported (Li PATTERNING MICROSTAMPING PATTERNING

et al 2003).
Stamp
One of the most commonly used photoresist is SU-8, Stencil

originally developed by IBM, and currently marketed by

MicroChem Corporation (Newton, MA, USA), and SOTEC
Channels for
Microsystems (Renens, Switzerland). This negative photo- microfluidics

resist is crosslinked upon exposure of near UV energy in the

range of 350 to 400 nm, and can be developed with a number
Material to
of substances including propyleneglycol monoether acetate, be stamped
ethyl acetate and diacetone alcohol. One of the main advan-
tages of SU-8 is that it permits generation of tall structures, of
Figure 2 Soft lithography includes the techniques of microfluidic patterning,
more than 1000 μm in height (Becker and Gärtner 2000). microstamping and stencil patterning. All three techniques are based on the
generation of the replica of a microstructure from a poly(dimethyl siloxane)
Photolithography has reached wide acceptance in the (PDMS) mold prepared through other microfabrication methods such as
field of microfabrication because of the high resolution and photolithography.

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Betancourt and Brannon-Peppas

Microstamping, also known as microcontact printing, Etching

is based on the construction of a stamp that has the desired Etching is a process that aims to create topographical features
pattern with the PDMS mold. Molecules to be transferred on a surface by selective removal of material through physical
are placed on the surface of the stamp and “printed” on or chemical means. Etching can be isotropic if it preceeds
a receiving surface upon stamping, thus forming a self- equally in all directions or anisotropic if it proceeds in one
assembled monolayer (SAM) (Voldman et al 1999; Chen specified direction. Figure 3 depicts these two etching mecha-
and Pepin 2001; Curtis and Wilkinson 2001). Depending nisms. As shown in Figure 3A, isotropic etching occurs not
on the application, peptides, proteins, polysaccharides and only in the direction of depth, but also laterally, and results
other molecules can be stamped. The stamped layer can in a curved profile. Anisotropic etching, shown in both
protect the substrate during etching or deposition proce- Figures 3B and 3C, occurs in only in one direction, usually
dures, which will be described in more detail later in this selectively increasing the depth of the cavity.
section. One great advantage of this microfabrication The mechanisms used for etching utilize liquid chemicals
method is that the stamp can be reused to make pattern or gaseous physico-chemical processes. These two methods
replicas. are more commonly known as wet etching or dry etching,
The second soft lithography technique, stencil patterning, respectively (Li et al 2003). As shown in Figures 3B and 3C,
creates templates by preventing PDMS from covering the dry anisotropic etching results in a flat profile, while wet
master template, as can be seen in Figure 2. The end result anisotropic etching results in cavities with inclined side-
of this process is a PDMS model with holes in the pattern of walls. The characteristic slanted profile of wet anisotropic
the master. Different methods can be used to prevent PDMS etching is a result of the interaction of the etching reagent
from covering the master features, such as placing plates with the crystalline structure of the material being etched.
against the master following PDMS addition, or adding the The crystal structure determines the rate of etching that
PDMS to a thickness smaller than that of the master features occurs at each crystal plane. For most applications, the flat
(Li et al 2003). profile of dry anisotropic etching is adequate. Reactive ion
Microfluidic patterning, the last of the soft lithographic
techniques, utilizes a PDMS mold to create microchan-
A B C
nels against a substrate. These microchannels can then be
used to pattern fluid materials onto a substrate (Li et al
2003). This technique has been utilized for the patterning
of cells in tissue engineering applications (Tan and Desai
2003). One important feature of these microchannels is
their ability to maintain separate fluid streams through a
single channel because of laminar flow (Andersson and
van den Berg 2004). This characteristic can be utilized for
Figure 3 Etching profiles generated with (A) isotropic etching, (B) dry anisotropic
the study of cellular response to the stimuli of different etching, and (C) wet anisotropic etching.
materials.
etching, which utilizes oxygen or fluorine plasma, has also
Film deposition been extensively used.
The application or growth of layers of materials, or films,
on the surface of microstructures is a common procedure of Bonding
microfabrication. Films can play a structural or functional Reversible and irreversible bonds can be formed between
role in the design. For example, they may be used during microstructures to form tight seals or to obtain desired
microfabrication as sacrificial or masking layers that structures. There are numerous bonding methods available
protect the base material from etching, or even as electrical that are specific for the material of interest. For example,
components for a microfabricated device. Numerous types irreversible anodic bonding is possible between a silicone
of materials are used for the generation of films. Among substrate and a non-pure glass film (Voldman et al 1999).
these, the most commonly used are plastics, silicon- The formation of this bond requires exposure of the system
containing compounds, metals, and biomolecules (Voldman to temperatures in the order of 400οC, high pressure, and an
et al 1999). electric field. Fusion bonding, on the other hand, consists of

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the annealing of two surfaces at high temperatures (∼1000oC) Focused ion beam lithography
(Voldman et al 1999). This type of lithographic technique utilizes ions in place
Bonding of polymers can be carried out through heating of electrons to pattern a resist. Ions are generated from a
above the glass transition temperature and applying pressure liquid metallic tip, containing elements such as gallium,
to seal the structures, through laser welding, or ultrasonic “filtered” to allow only one type of ion to interact with the
welding (Becker 2000). In addition, adhesives can also be resist, and focused on the material surface with electrostatic
utilized to bind two materials. However, the addition of lenses (Chen and Pepin 2001). Common operational energy
intermediate layers will affect the properties of the system levels are in the range of 10 to 200 keV. Focused ion beam
and must be taken into account. lithography can also be used to pattern features directly
to a substrate – without the need for a photomask – by
Nanofabrication techniques either selective material removal or deposition (Gates et al
Nanofabrication utilizes principles similar to those of 2005). Compared with electron ion beam lithography, the
microfabrication for the generation of patterns or devices at patterning speed offered by this technique is significantly
the nanoscale level, ie, of sizes ranging from 1 to 100 nm. slower because of lower achievable ion current density, when
Some authors consider sizes of up to 1000 nm to be within the compared with that of electrons. Common feature resolutions
realm of nanostructures. Various microfabrication techniques are in the range of 20 nm, with a minimal 5 nm lateral feature
have been utilized to achieve features within this range. Soft size (Gates et al 2005).
lithographic techniques, for example, have been employed
for the production of features with a resolution of less than Colloid monolayer lithography
200 nm through the use of materials stiffer than PDMS for This lithographic method utilizes self-organized one- or
the fabrication of the stamp (Chen and Pepin 2001). Features two-dimensional colloidal systems as layers for nanofab-
of less than 40 nm have been produced with conventional rication. This technique is an economical alternative to the
photolithography utilizing light of 193-nm wavelength (Gates common electron or ionic lithographic methods, but is still
et al 2005). In addition, a number of special lithographic able to produce patterns at the nanoscale. Colloidal mono-
techniques have been developed to accomplish this miniatur- layers can be generated through a number of self-assembly
ization and will be discussed in the following sections. processes. For example, colloidal particles can be deposited
on the surface of a substrate in solution prior to solvent
Electron beam lithography evaporation, through spin coating, or through electrophoresis
Electron beam lithography is the principal nanofabrication (Burmeister et al 1999).
technique used to create features at the nanoscale level on a If the size and geometry of the colloidal particles is
material. This technique utilizes an electron beam to scan a precisely controlled, it is in theory possible to also control
material and form the desired pattern. Magnetic lenses are the spatial distribution of the colloids. For example, mono-
used to focus the beam. Commonly used electron sources are dispersed spherical particles can be close-packed into
thermoionic emitters and thermal field emitters which have hexagonally arranged monolayers, ie the conformation of
outputs in the range of 1 to 200 keV, but are most commonly highest density for this geometry (Burmeister et al 1999).
used in the range of 50–100 keV (Chen and Pepin 2001). The Nonetheless, a number of parameters including colloid
resolution obtained through this type of lithography is greatly concentration, solvent evaporation rate, wetting character-
influenced by the beam spot size. Specimen position and istics of the substrate, and competition with multiple-layer
beam characteristics are electronically controlled to achieve formation influence the resulting array. It has been reported
the desired nanoscale resolution. Various researchers have that the achievable resolution offered by this technique,
demonstrated resolutions on the order of less than 10 nm. when the colloidal monolayer is utilized as a resist, can
Chen et al (1996) demonstrated the feasibility of producing be as low as 5 nm in all three dimensions (Curtis and
5 to 7 nm-wide etched lines on a silicon substrate by pattern- Wilkinson 2001).
ing a polymethylmethacrylate photoresist with an electron These colloid monolayers can be used as a protective
beam of less than 5 nm in diameter at a voltage of 80 kV barrier against etching and, consequently, transfer their semi-
(Chen and Ahmed 1993). The main drawback of electron random distribution pattern to the substrate material (Curtis
beam lithography is the cost associated with purchase and and Wilkinson 2001). More specifically, these etch masks
maintenance of the system (Gates et al 2005). would result in the removal of the substrate material located

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Betancourt and Brannon-Peppas

below the colloid interstices. The same analogy can be made biomolecules such as proteins (Decher 1997; Lvov et al 1998)
for applications in selective film deposition. Depending on and DNA (Taton et al 2000). This technique also provides
the application, the colloidal particles can be either removed great control over the film structure, thickness, and function.
or left in place. In addition, because film formation occurs by adsorption
Another application of colloidal monolayers has been of polyanions or polycations from solution, the number of
recently reported by the group of Dr. Saochen Chen at the possible morphologies and sizes are only limited by those
University of Texas at Austin. In their design, colloidal of the substrate to be coated.
silica spheres were deposited on the surface of a poly
(ε-caprolactone) film. After self-arrangement of the spheres, Electrically induced nanopatterning
a laser beam was used to irradiate the samples. The spheres, Electrically induced nanopatterning techniques utilize
which have diameters larger than the wavelength of the electrostatic interactions between a thin dielectric material
light, act as lenses, thus intensifying the effect of the laser liquid film and an electric field gradient to produce lateral
beam on the substrate material (Lu and Chen 2004). Upon patterns and structures at the nanometer scale (Schäffer et al
disappearance of the spheres due to laser action, holes 2000). The nanofabrication system is analogous to a capacitor
arranged in the original sphere pattern are left behind. Thus, because it consists of two parallel electrodes separated by an
this technique can be used for high throughput patterning air gap of less than a micron, as shown in Figure 4. A polymer,
of nanoholes. such as polystyrene, is applied to one of the electrodes by
spin coating. Upon exposure to high temperature (above the
Molecular self assembly glass transition temperature of the polymer) and to an electric
Molecular self assembly is an alternative to lithographic field generated by the voltage across the electrodes, electro-
techniques for the fabrication of features and structures at the static forces develop and result in the destabilization of the
nanometer scale. It is based on thermodynamically favored polymeric film. Since the instabilities have a defined periodic
interactions of molecules such as peptides, proteins, and undulation pattern, this system ultimately results in the
DNA, and other organic or inorganic molecules (Rajagopal formation of polymeric columns at the location of the peaks
and Schneider 2004). Molecules are spontaneously brought of the polymer “wave”, ie, those locations with the highest
together to energetically stable conformations favored by polymer thickness or smallest air gap between the polymer
noncovalent forces including hydrophobic, van der Waals, and the top electrode (Schäffer et al 2000, Liu et al 2003).
and electrostatic interactions, as well as hydrogen bonding. Lateral column density and order can be altered by changing
This technique has a number of advantages including the parameters such as the initial polymer film thickness, or the
ability to fabricate three-dimensional structures and the inter-electrode spacing (Schäffer et al 2000).
potential for molecular control of the material (Rajadopal If in addition the electrode opposite to the polymer
and Schneider 2004). Molecular self assembly is able to possesses microstructured patterns, the instabilities will
control pattern formation to the sub-nanometer scale. Self first develop at the locations where the distance between
assembly of monolayers of amphiphilic peptide β-hairpins the electrodes is minimal because of the increased electro-
was shown to result in organized structures containing static driving force. The result of these instabilities is that
2.5 nm hairpins spaced by less than 0.3 nm (Rajagopal and the polymer extends across the air gap toward the opposite
Schneider 2004). electrode, thus generating nanometer scale “columns” in the
Layer-by-layer assembly, one of the self assembly specific pattern of the top electrode (Schäffer et al 2000), as
methods, is based on the consecutive deposition of multiple can be seen in Figure 4. These columns are then solidified
thin polyion films from solution utilizing the electrostatic by cooling below the polymer’s glass transition temperature.
attraction that develops between oppositely charged molecules Pattern reproductions with features of 140 nm were reported,
as a driving force for assembly (Decher 1997). Time for but the authors suggest that generation of features of less
adsorption of polyions onto surfaces ranges from minutes than 100 nm is feasible.
to hours depending on factors such as polyion type and This technique has further been utilized to generate
concentration. Intermediate washes between sequential porous templates for the fabrication of nanowire arrays with
depositions are performed to avoid contamination from very high densities (Thurn-Albrecht et al 2000). This system
previous solutions. This technique has been widely accepted utilized a film of the diblock copolymer of polystyrene (PS)
because it permits the use of numerous materials, including and polymethylmethacrylate (PMMA) that self-assemble into

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 Micro- and nanofabrication in biomedical devices

A Top B were used for rapid fabrication of functional structures. The

Patterned
Electrode
systems fabricated incorporated highly organized two- and
three-dimensional arrangements of monodispersed pores,
Voltage
Source δ which could also be patterned for the formation of larger
V
structures. In addition, different molecules were success-
Instability fully added for functionality; some of the molecules utilized
Polymer develops
Bottom where δ is included mercaptopropyltrimethoxysilanes for noble metal
Electrode smallest
coupling, and aminopropyltrimethoxysilanes for metal, dye,
Figure 4 Schematic of electrically-induced nanopatterning process. (A) The or bioactive molecule coupling (Fan et al 2000).
system utilized for electrically induced micropatterning consists of two electrodes
separated by an air gap of thickness δ. A thin film of a polymer to be molded is
applied to the bottom electrode. Upon exposure of an external magnetic field,
electrostatic forces surpass surface tension forces, and instabilities develop on the
X-ray lithography
polymer at the sites where δ is smallest. (B) Columns formed at the sites of the This technique employs electromagnetic radiation of
major instabilities mimic the pattern of the top electrode. Based on a figure from
Schäffer et al (2000). wavelengths in the range of 0.5 to 4 nm, commonly known
as soft X-rays, for pattern transfer from a mask to a substrate
material (Chen and Pepin 2001). Common sources of soft
arrays of PMMA cylinders surrounded by a PS matrix. This X-rays are synchrotrons or laser-induced plasma generators
film was coated onto a bottom electrode, and exposed to an (Chen and Pepin 2001). X-ray masks are usually in the order
electric field. The material migrated toward the top electrode of a few microns in thickness, and are usually composed of
maintaining the self-assembled domains and, after cooling, silicon carbide. Absorber patterns, analogous to the opaque
was subjected to deep ultraviolet irradiation to crosslink the regions of a photolithographic mask, are created with
PS matrix and degrade the PMMA columns, which were heavy metals such as gold, tungsten or tantalum (Chen and
then removed to form the porous template. Electroplating Pepin 2001).
was later used to fill the nanopores with metals to fabricate In this technique, it is possible to perform lithography
nanowire arrays. with a mask-to-wafer distance of several microns. However,
larger gap distances proportionally reduce the achievable
Rapid prototyping resolution (Chen and Rousseaux 1996; Chen and Pepin
Rapid prototyping combines various nanofabrication 2001). Additional parameters that influence the resolution
techniques for the generation of complex geometrical patterns, offered by this technique are Fresnel diffraction, also known
multi-layered structures, and structures with chemical func- as near-field diffraction, and photoelectron diffusion into
tionality (Fan et al 2000; Lu and Chen 2004). This technique resist films, or photoelectron blur (Chen and Rousseaux 1996;
offers advantages over common lithographic methods such Chen and Pepin 2001). Unfortunately, the Fresnel diffraction
as resolution of features below 100 nm, the ability to include is proportional to the wavelength of energy used, while the
diverse functionalities to the materials, and most importantly, photoelectron blur is inversely proportional. Consequently,
the reduction of fabrication time from hours to seconds (Fan to achieve patterns with small resolutions it is necessary to
et al 2000). In general, computer assisted design (CAD) is balance these two variables. Resolutions of less than 30 nm
utilized to control the fabrication process. Some of the fabri- have been reported (Chen and Pepin 2001).
cation methods that are utilized in rapid prototyping include
direct deposition, three-dimensional printing, selective laser Ion projection lithography
sintering, and laser stereolithography (Lu and Chen 2004; Ion projection lithography is based on the exposure of a
Yeong et al 2004). These techniques generate micro and nano- wafer to hydrogen or helium ions (Chen and Pepin 2001).
structures in a layer-by-layer approach. Rapid prototyping As in photolithography, ion projection lithography uses a
techniques have been studied for the fabrication of scaffolds mask to prevent exposure of part of the substrate to ions.
for tissue engineering (Yeong et al 2004). In this case, however, the masks contain an ion absorbing
The utilization of rapid prototyping techniques was material that prevents ion projection to the substrate under-
illustrated by a collaborating group from the University neath the absorbing pattern. Common ion energies range
of Mexico and Sandia National Laboratories (Fan et al from 70 to 150 keV (Chen and Pepin 2001). Fabricated
2000). In this work, evaporation-induced self assembly, features of the order of 50 nm have been reported (Hirscher
pen lithography, ink-jet printing and dip-coating techniques et al 2002).

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Applications silicon substrate and the metal layers, the internal stress on the
Micro- and nanofabrication techniques have enabled the metal causes it to roll into cylindrical configuration. Cylinders
scientific and medical community to expand the applications as small as 1.5 microns in diameter and 5 microns in length,
of already-existing devices through miniaturization, and to with walls of tens of nm in thickness were reported.
create completely new devices with use of the increased con- Multilayered nanoparticles prepared by atom-by-atom
trol of size, morphology, topology, and functionality offered or layer-by-layer self assembly for the delivery of drugs or
by these techniques. These novel micro- and nanodevices genes have been developed (Prow et al 2004). These systems
have been able to contribute immensely to the fields of cell offer the possibility to combine multiple sequential functional
biology, molecular biotechnology, and medicine. It is now layers that guide the particles through the drug delivery
possible to study the interactions of biomaterials with biologi- process one layer at a time. For example, sequential layers
cal systems at the cellular and molecular scale, and to design can be loaded with targeting molecules, membrane entrance
new synthetic systems that are able to alter physiological molecules, intracellular targeting molecules, and active
responses by capitalizing on these findings. Applications of agents such as drugs and genes for targeted intracellular
micro and nanodevices in the medical and pharmaceutical delivery (Prow et al 2004).
field are the focus of this section.
Gene delivery with micro
Drug delivery devices and nano-machined devices
Micro- and nanofabrication techniques offer a range of A number of micro and nanodevices have been used for the
possibilities for the preparation of peptide, protein, drug, or delivery of genes to target cells. One such system utilizes
gene delivery devices. The ability to control the size, architec- “micromechanical piercing” or microprobe elements to
ture, topography, and functionality of drug delivery vehicles deliver genes coated onto their surface through cell penetra-
could result in the fabrication of systems that behave in highly tion (Reed et al 1998). Microprobes of 80 microns in height
predictable manner both in vitro and in vivo, thus surpassing and with a sharp point of less than 200 nm in diameter were
the capabilities of current drug delivery systems. reported. Successful expression of genes delivered with
microprobes was demonstrated in plant, animal, and mam-
Injectable micro- and nanodevices malian cells (Reed et al 1998; Hashmi et al 1995). This
The fabrication of injectable self-assembled micro reservoirs system as described is only practical as a research tool for
for controlled drug delivery was recently reported (Pizzi the transfection of cell monolayers; however, application
et al 2004). The design of these micro reservoirs consists of of the concept for in vivo therapeutic purposes is being
metallic cylindrical containers within which the drug was investigated.
loaded. The metallic cylinders, made of biocompatible met-
als such as titanium or gold, were capped with degradable or Stents for drug delivery
non-degradable temperature sensitive polymeric membranes Reed and colleagues developed microfabricated intravascular
on both ends. These membranes could either degrade or stents for the treatment of restenosis (Reed et al 1998). The
become more permeable at high temperatures. Drug release design is targeted at increasing the efficacy of pharmaceutical
could then be controlled externally through the application of prevention of restenosis in comparison to local administration
electromagnetic radiation at the site of pathology. Therapeu- regimes that are unable to cross the internal elastic lamina and
tic effect was a result of the synergistic combination of drug compressed plaque that are present in pathological vessels.
delivery at the site of pathology, and heating of the metallic These stents incorporate microprobes, which upon catheter-
walls of the micro reservoirs above viable temperatures upon based localized deployment are able to perforate compressed
application of the electromagnetic radiation. Microfabrica- plaque and internal elastic lamina, and deliver anti-restenosis
tion of this system is achieved through deposition of two agents or therapeutic genes, which are previously coated
metal layers onto a flat silicon substrate and sacrificial layer onto the microprobe surfaces, directly into coronary tissue.
by thermal evaporation deposition. Drugs are immobilized Fabrication of microprobe arrays for ex vivo study of
onto the exposed metallic surface either chemically or physi- barrier penetration in rabbit models was carried out through
cally. Photolithography and wet etching techniques are then oxidation, photolithography and anisotropic etching of a
employed to form a large number of independent squared silicon substrate (Reed et al 1998), techniques that result in
elements. Upon etching of the sacrificial layer between the planar structures. In order for this system to be feasible for

490 International Journal of Nanomedicine 2006:1(4)

 Micro- and nanofabrication in biomedical devices

in vivo applications, the microprobe-containing stents require patterned into dots of fluorine and oxygen plasma to etch
cylindrical geometry and the ability for deformation during the uncovered surfaces in such a way that microneedles
balloon-based inflation (Reed et al 1998). The authors discuss are formed on the regions protected by the mask (Henry
the use novel anodic oxide microfabrication to prepare struc- et al 1998). Hollow silicon microneedles are prepared by
tures with high aspect ratio which could be used to solve first etching a silicon substrate to form holes or conduits,
some of these problems (Reed et al 1998). followed by the previously described procedure to form the
microneedle body (McAllister et al 2003). Micromolding
Microneedles for transdermal drug delivery techniques, in conjunction with electrodeposition or
Microneedles have been developed for transdermal microinjection molding have been used for the fabrication
administration of proteins, drugs or genes with therapeu- of metal or polymer microneedles, respectively (McAllister
tic purposes. Transdermal administration of active agents et al 2003). Microneedles with various tip geometries were
offers advantages when compared to intravenous or oral also reported.
delivery routes. Intravenous administration is associated As a proof-of-concept study, solid silicon microneedles
with low patient compliance because of pain caused by the of 150 μm in height and less than 1 μm in tip radius of
injection. Oral drug delivery, on the other hand, is hindered curvature were tested on human epidermis ex vivo. Results
by the degradation of the active agent in the gastro-intestinal showed that microneedle arrays produced by this method
track, low bioavailability because of the difficulties asso- could be easily inserted, removed, and reinserted from
ciated with intestinal absorption, and the sequestration of the skin without significant damage, and resulted in up to
much of the drug upon absorption by the liver because 25 000-fold increase in permeability of calcein as a model
of the first pass effect (Lee and Yamamoto 1989). These drug (Henry et al 1998). Later ex vivo studies on human
problems are most important when the active agent is a epidermis showed that the permeability to calcein, insulin,
biomolecule such as a protein or DNA. Current transdermal bovine serum albumin, and even polystyrene microspheres
delivery systems, however, achieve low bioavailability of 50 μm diameter was increased by more than one or two
because of the low permeability of the stratum corneum, orders of magnitude when solid microneedles were either
a layer of dead cells of 10–15 μm in thickness that acts inserted, or inserted and removed, respectively (McAllister
as a barrier. Numerous chemical and physical methods et al 2003). In vitro studies on DU145 cells showed cells
have been attempted with the purpose of increasing skin could uptake calcein administered with solid microneedles
permeability for drug delivery purposes, including chemical with only about 10% cell mortality (McAllister et al 2003).
enhancers, iontophoresis, electroporation, and ultrasound Finally, in vivo studies of transdermal delivery with hollow
permeabilization (Bommannan et al 1992; Fang et al 1998; microneedles in diabetic hairless rats showed that microinjec-
Lombry et al 2000; Pillai and Panchagnula 2003; Kalia et al tion of insulin resulted in a statistically significant reduction
2004). Microneedles offer an alternative to conventional of the blood sugar level for a period of 5 hours. In addition,
transdermal delivery and to permeabilization techniques in vivo use of solid microneedle arrays on human volunteers
because they act as channels that transport the drug across suggested that application was not painful and did not cause
the stratum corneum barrier into the deeper tissue (dermis) irritation (Henry et al 1998).
where it can enter the systemic circulation. In addition, by Different microneedle designs have been utilized by other
careful control of the microneedle mechanical strength groups for the delivery of the antisense oligonucleotides
and length, it is possible to deliver drugs across the dermal (Lin et al 2001), proteins (Matriano et al 2002), and plasmid
barrier while evading the nerves, thus resulting in painless DNA (Mikszta et al 2002; Chabri et al 2004). The fabrica-
administration (Henry et al 1998; Kaushik et al 2001; Tao tion of biodegradable microneedles has also been proposed
and Desai 2003). (Park et al 2005). Figure 5 shows examples of solid silicon
Microneedles have been fabricated out of a range of microneedles fabricated by reactive ion etching.
materials including silicon, metals, polymers, and glass Recently, emphasis has been placed on the fabrication
(McAllister et al 2003). Reactive ion etching has been and utilization of devices for enhanced microneedle drug
widely used for the production of solid and hollow silicon delivery. For example, Zahn and colleagues have developed
microneedles (Henry et al 1998; McAllister et al 2003). an on-chip planar positive displacement pump for portable
Here, solid silicon microneedle arrays were produced continuous drug delivery with microfabrication techniques
by exposing a substrate covered by a chromium mask (Zahn et al 2004).

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Betancourt and Brannon-Peppas

heparin, and human growth hormone at different time

intervals. Fabrication of these implantable devices utilized
compression-molding for generation of the reservoirs on a
PLLA disk, and microinjection for generation of reservoir
membranes and drug filling (Richards et al 2003). Each
11-mm device had 36 reservoirs with a capacity of more
than 120 nL each.
Low and colleagues have also developed responsive,
reversible polymeric valves based on hydrogel actuators
for microchip delivery of drugs in response to physiological
Figure 5 Scanning electron microscopy image of relatively short solid silicon changes (Low et al 2000). Such system would deliver
microneedles (25 μm in height) prepared by reactive ion etching. These
nanoparticles were designed for cutaneous gene delivery. Reproduced with active agents only when needed, thus avoiding unnecessary
permission from McAllister D, Wang P, Davis S, et al. 2003. Microfabricated needles or possibly hazardous release of drugs which could occur
for transdermal delivery of macromolecules and nanoparticles: fabrication methods
and transport studies. Proc Natl Acad Sci U S A, 100:13755-60. Copyright © 2003 from ‘pre-programmed” microchips, such as those described
National Academy of Sciences, U.S.A. above.

Implantable microfabricated drug delivery chips Microfabricated bio- and muco-adhesive systems
Implantable microchips have been developed for on-demand Various alternative techniques have been investigated for
pulsatile release of a number of different drugs in varied improved oral delivery of drugs and biomolecules which
doses by the Langer group (Santini et al 1999; Langer and currently offers only limited bioavailability as a result of
Peppas 2003). The design consists of the microfabrication the properties of the gastro intestinal track: low stomach
of reservoirs within silicon substrates in which the drug is pH and presence of proteases that degrade active agents,
immobilized in either liquid or solid form (Santini et al 1999). and poor intestinal absorption of macromolecules. Bio- and
These reservoirs are covered by a membrane of a material muco-adhesive systems offer the possibility to increase
that can dissolve upon exposure to an electrochemical stimu- the residence time of drugs or drug carriers at the site
lus, such as gold. The microchip contains a cathode made of absorption, thus increasing the possibility that these
of silicon nitride or silicon dioxide (Santini et al 1999). An drugs are able to enter the systemic circulation and exert
electrical potential can be established between this cathode a therapeutic effect. Micro- and nanofabrication offer
and one of the reservoir membranes which act as anodes. This the opportunity to design and produce oral drug delivery
potential triggers dissolution of the membrane, and results systems with high degree of functionality for bioadhesive
in the pulsatile release of the drug. purposes.
The implantable microreservoirs were generated by One such system utilizes poly(methacrylate) micropar-
sequential microfabrication techniques including UV ticles for bioadhesion. These microparticles were fabricated
photolithography, chemical vapor deposition, electron beam by lithography and reactive ion etching (Tao and Desai 2003),
evaporation and reactive ion etching (Santini et al 1999). The and were surface modified with amine groups to which
model microchips had a size of 17 mm on each side, and con- biological molecules, such as avidin-lectin complexes, could
tained approximately 1000 reservoirs, each of which could be attached. Lectins were chosen because they are known
contain a volume of up to 25 nL (Santini et al 1999). to bind glycoconjugates on cell surfaces with great affinity.
More recently, a biodegradable microchip system fab- The microparticle square morphology and size (150 μm wide
ricated from poly(lactic-co-glycolic acid)-based polymers by 3 μm in thickness) were chosen in order to maximize
was developed for long term pulsatile delivery of drugs the surface area in contact with the intestinal walls. This
(Richards et al 2003). Specifically, the main body of the system was found to significantly increase binding to Caco-2
device which contains reservoirs for drugs was made from monolayers.
poly(L-lactic acid) (PLLA), while the membranes covering Other groups have proposed the use of micro- and
these reservoirs were prepared from the fast-degrading nanofabrication techniques for the creation of patterned
polymer poly(D,L-lactic-co-glycolic acid) (PLGA). mucoadhesive structures based on the interactions between
Using PLGA of different molecular weight for membrane mucin, ie, the main organic component of intestinal
preparation, they were able to show release of dextran, mucus, and hydrophilic polymers (Kim and Peppas 2003;

492 International Journal of Nanomedicine 2006:1(4)

 Micro- and nanofabrication in biomedical devices

Peppas and Huang 2004). Lithographic photopolymeriza- on the cantilever that results in a specific degree of bending.
tion of hydrogels onto silicon substrates was proposed for The sensitivity of these systems was observed to be of up
these systems. to 1 nm per 5 × 10−5 pH units, with maximum sensitivity
between pH 5.9 and 6.5 (Bashir et al 2002; Hilt et al 2003).
Micro and nanofabricated biosensors Lei et al reported a similar system in which hydrogels were
Sensors consist of devices that detect and/or measure a patterned onto microcantilevers through photolithography
specific compound or condition in their environment, and and dry etching, instead of through polymerization at the sur-
generate a corresponding output through the action of a face of the microcantilever (Lei et al 2004). This alternative
transducer. Depending on the design, micro- and nanosensors method avoids the use of photoinitiators that could possibly
are able to identify changes in pressure, temperature, ionic impose limitations on the in vivo use of these systems.
strength, or concentration or a target molecule, just like their A similar system was designed for sensing of glucose
macro-metric counterparts. through the immobilization of the enzyme glucose oxidase
Microcantilevers have been shown in the past to work (GOx) onto gold-coated silicon cantilevers. Enzymatic reac-
as very sensitive transducers for sensing applications. Their tion of glucose in solution with the immobilized GOx results
function is based on the generation of surface stresses, and a in a measurable cantilever deflection that is representative of
subsequent bending of the cantilever in response to changes the concentration of glucose in the medium (Pei et al 2004).
in environmental conditions, or upon binding of a target This system was shown to be effective over a wide range of
molecule. glucose concentrations up to 20 mM.
Microcantilevers functionalized with pH-sensitive hydro- Biosensors based on configurational biomimetic imprinted
gels were recently developed by the group of Dr Nicholas hydrogels with biorecognitive properties have also been
Peppas (Bashir et al 2002; Hilt et al 2003). The system proposed (Byrne et al 2002; Hilt and Byrne 2004). Three-
shown in Figure 6 is an example of these environmentally dimensional biomimetic imprinted polymer networks are gener-
sensitive cantilevers. These pH-sensitive hydrogels, in this ated by self assembly of polymers and/or functional monomers
case prepared by selective crosslinking of poly(methacrylic around target molecules during the formation of the hydrogels.
acid) and poly(ethylene glycol) dimethacrylate, exhibit a Self assembly is driven by thermodynamically favored chemical
swelling behavior that responds to changes in the pH of or biological interactions (except covalent bonding) between
the surrounding medium. Hydrogels were patterned by the target molecule and the hydrogel components. Upon
photolitography onto micro-machined silicon cantilevers removal of the target molecules from these hydrogels, cavities
by selective UV free-radical polymerization. An organosi- with biorecognitive properties are left behind. Applications of
lane compound was used to form covalent bonds between biomimetic materials include biosensors that are able to recog-
the silicon substrate and the hydrogel polymers. Upon pH nize a target molecule, and deliver drugs or remove unwanted
changes, the resulting hydrogel swelling creates surface stress compounds for therapeutic purposes (Hilt and Byrne 2004).

Silicon Polymer
Cantilever Cantilever I
B

d A Cantilever II

Cantilever III

200 µm

Oxide

Silicon

Figure 6 Schematic (left) and image captured with a Microscope II in Nomarski mode (right) of silicon cantilevers patterned with photolithography with
environmentally sensitive hydrogels. Swelling of the hydrogel as a result of pH changes results in pH-dependent deflection that can be quantified based on the differences of
focus planes A and B. The thickness of the patterned hydrogels was determined to be of approximately 2.5 μm. Unpublished images provided by Dr. Nicholas Peppas.

International Journal of Nanomedicine 2006:1(4) 493

Betancourt and Brannon-Peppas

Conclusions Fang JY, Lin HH, Chen HI, et al. 1998. Development and evaluation on
transdermal delivery of enoxacin via chemical enhancers and physical
Adaptation of micro- and nanofabrication techniques iontophoresis. J Control Release, 54:293-304.
derived from the semiconductor industry has led to the Gates BD, Xu Q, Stewart M, et al. 2005. New approaches to nanofabrication:
molding, printing and other techniques. Chem Rev, 105:1171-96.
creation of novel devices for use in the medical and Hashmi S, Ling P, Hashmi G, et al. 1995. Genetic transformation of
pharmaceutical fields. These systems promise to offer nematodes using arrays of micromechanical piercing structures.
improved characteristics including enhanced control of Biotechniques, 19:766-70.
Henry S, McAllister DV, Allen MG, et al. 1998. Microfabricated
feature geometry, size and complexity, feasible mass microneedles: a novel approach to transdermal drug delivery. J Pharm
production, portability, and miniaturization. In addition, Sci, 87:922-5.
Hilt J, Gupta A, Bashir R, et al. 2003. Ultrasensitive biomems sensors
because these techniques enable the production of devices based on microcantilevers patterned with environmentally responsive
at the cellular and sub-cellular levels, they open the doors hydrogels. Biomed Microdevices, 5:177-84.
to the creation of new strategies for the study and manipu- Hilt JZ, Byrne ME. 2004. Configurational biomimesis in drug delivery:
molecular imprinting of biologically significant molecules. Adv Drug
lation of molecules, cells, and tissues, thus providing new Del Rev, 56:1599-620.
avenues for the investigation of pathological mechanisms Hirscher S, Kummel M, Kirch O, et al. 2002. Ion projection lithography
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and novel treatment options. This paper describes some of Engineering, 61-62:301-7.
the main micro- and nanofabrication techniques that have Kalia YN, Naik A, Garrison J, et al. 2004. Iontophoretic drug delivery. Adv
been published in the literature and examples of how these Drug Del Rev, 56:619-58.
Kaushik S, Hord AH, Denson DD, et al. 2001. Lack of pain associated with
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and diagnostics. Kim B, Peppas NA. 2003. Poly(ethylene glycol)-containing hydro-
gels for oral protein delivery applications. Biomedical Microdevices,
5:333-41.
Langer R, Peppas NA. 2003. Advances in biomaterials, drug delivery, and
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