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Understanding nanoscale
drug delivery systems
by Udo Bakowsky* and Johannes Sitterberg
*Corresponding author:
Prof. Dr. Udo Bakowsky, Institut für Pharmazeutische Technologie
und Biopharmazie,
Philipps Universität Marburg, Ketzerbach 63, D-35032 Marburg,
E-mail: bakowsky@staff.uni-marburg.de
Introduction
The development of new drugs with short biological half-life
and highly active drugs with potentially dangerous side effects
has led to an increased interest in techniques to deliver
these drugs to their desired site of action. New high affinity
ligands often lack the biopharmaceutical properties that
are necessary to use the compound as a drug, e.g., solubility
or permeability. In addition, the interaction between the
administered drug and the human body may still result in
short circulation time, due to rapid clearance by the reticulo-endothelial
system or fast metabolism. These novel drug molecules demand
new formulations which protect the drug, as well as the host
and are able to direct the drug to the destined organ. One
possibility to overcome these problems is the development
of nanoscale carriers for various applications, such for
a nasal or pulmonary administration.
Controlled drug delivery offers multiple options for the optimisation
of drug action by adjustments of the release rate, design of
pro-drugs and the alterations in the accumulation of the drugs
at their desired site of action (drug targeting). Drug targeting
can be achieved by incorporation of the drug into polymeric
particles or liposomes, use of solid lipid nanoparticles, surfactant-
or lipid-modified hydrogels or systems based on biodegradable
nanoparticles. Additionally to these rather unspecific controlled
release systems, the affinity towards the site of action (bioadhesion)
can be further enhanced by modification of the surfaces with
target-seeking moieties, such as lectins, antibodies, peptides,
carbohydrates or invasion factors [1-13]. In this rapidly growing
area of research, special attention has been paid to the physicochemical
characterisation of colloidal drug carrier systems. This includes
the determination of the size distribution, charge density,
calorimetry, time dependence of the drug release and analysis
of the adhesive properties using e.g., quartz crystal micobalance
or surface plasmon spectroscopy. For the visualisation of the
nano-carriers, scanning force microscopy and electron microscopy
could be used.
Visualisation of drug delivery systems
The visualisation of surface morphology allows an understanding
of physical, chemical and biological phenomena. For many
years, optical microscopy has been used as a tool to produce
images of surfaces. The resolution that can be achieved by
traditional light microscopes is limited to approximately
one micrometer by the Nyquist relation, and measurements
in the z-direction are not possible. Developed in the 1940’s,
the next most widely used instrument for investigating surface
morphology has been the scanning electron microscope (SEM).
With this technique, only the near surface of samples can
be visualised. Similar to an analysis by optical microscopy,
SEM only measures in the x and y dimension of a sample and
insight into the z-direction can not be obtained. With today’s
SEM, resolution is limited to about five nanometres, owing
to the properties of the electromagnetic lenses. However,
this resolution can only be achieved under vacuum conditions.
Furthermore, a rather laborious sample preparation is often
required for SEM, as it may include steps, such as freeze
drying, staining or metal coating. Scanning force microscopy
(SFM), also known as atomic force microscopy (AFM), was developed
by Binnig and co-workers in 1986 [14]. SFM allows the imaging
of conducting, as well as non-conducting samples. High lateral
and vertical resolutions can be achieved in vacuum, in air
and even on liquid-covered surfaces. Measurements under physiological
conditions are also possible. In addition, with this microscopic
approach, it is not only possible to analyse the topography
of a sample, but also other physical properties, including
friction forces, softness and viscoelasticity, and the charge
density on a nanometre scale. With the optimal choice of
equipment, resolutions in the range between 0.1 nm and 2.0
nm for the x-y dimension, and less than 0.1 nm for the z-dimension
can routinely be obtained. All these options have made SFM
a very useful tool in many biological, medical or pharmaceutical
applications. Large objects, such as whole microparticles
can be imaged, as well as smaller structures in a nanometre
range.
The principle of scanning force microscopy
The main element of every SFM is the tip-cantilever system
(figure 1). The cantilever can be made from different materials,
usually silicon or siliconnitride. The choice of material
depends on the specific application. A sharp tip is mounted
on one end of a 100 to 500 µm long lever. The geometry
of this tip is crucial, as it represents one of the major
parameters that determine the resolution of a measurement.
The highest resolution can be achieved with a tip that tapers
off in a single atom. Imaging of a conventional SFM tip reveals
it to be spherically shaped with a diameter approximately
5 nm.
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| Figure 1: Schematic
of a scanning force microscope (SFM). The cantilever /
tip system is the “heart” of the SFM and determines
the resolution and the quality of the measurements. |
In order to be moved over the sample surface, the tip-cantilever
system is integrated into a stylus profilometer. Such equipment
has been used in the optics industry for many years. In essence,
a stylus profilometer is a piezoelectric scanner that can
generate movements of an accuracy and magnitude required
to generate topographic images with a resolution of some
nanometres. The movement of the piezoelectric elements
can be controlled by the voltage that is applied across
its electrodes. Depending on the design of the SFM, the
scanner is used either to move the sample underneath the
cantilever or to move the cantilever over the sample. This
feature of the equipment is the limiting factor for the
maximum scanning field. Hence, distances of about 125 microns
in the x-y direction and 10 microns in z-direction can
be covered. This is sufficient for many but not all technological
or biological purposes. When the cantilever is moved over
a structured surface, the attractive and repulsive forces
between tip and surface will change. These forces are measured
by sensing the deflections of the cantilever. Deflections
of the cantilever can be detected by a variety of methods,
such as electron interferometry, optical deflection or
capacity methods. In SFM, a number of different scanning
modes can be distinguished that differ primarily in the
type of interaction between tip and sample that is measured,
but also, at least in some cases, in the way the tip is moved
across the surface. The most relevant techniques for technological
and biological applications are contact SFM, TappingModeTM
SFM, lateral force microscopy and force spectroscopy.
Scanning force microscopy techniques
When using the contact technique, the tip is always in contact
with the surface and the cantilever scans line by line across
the sample. Accordingly, the topography of the sample results
in deflections of the cantilever which are detected and amplified
in order to produce an image. This image shows a map of tip-sample
interactions resulting from the inter-atomic repulsive forces
between these two surfaces. Especially, when scanning soft
material, the surface can be damaged and the possibility of
monitoring artefacts has to be taken into account.
TappingModeTM or intermitted contact imaging was a key advancement
in SFM of soft, adhesive or fragile samples, i.e., most biological
materials. For tapping mode, the cantilever has to be oscillated,
close to its own resonance frequency. This can usually be achieved
with a piezoelectric element. The oscillating tip is then moved
toward the surface until it senses or “taps” the
surface. This reduction in oscillation amplitude is used to
identify and measure surface features.
Force spectroscopy can be used to measure forces between the
tip and the sample surface and to generate force-distance curves.
This type of operations can be divided into scanning techniques,
lateral force microscopy and force spectroscopy, and relies
on the measurement of forces at different points of the sample,
which thus allows one to construct force vs. distance curves.
By sensing the deflection of the cantilever, it is also possible
to monitor frictional interactions between the tip and the
sample. The contrast of SFM images generally depend the mechanical
properties of the surface and the probe, such as adhesiveness
and elasticity. Studies on the interactive forces between single
molecules have contributed significantly to our understanding
of major processes in nature. For example, the binding force
of complementary molecules (ligand-receptor pairs and drug-substrate
pairs) can be characterised by interpretation of force distance
curves in the pico-newton range.
Sample preparation
A proper sample preparation and the selection of a suitable
substrate for the specific sample are crucial for high resolution
and artefact-free scanning force microscopy. The substrate
for high resolution imaging have to be flat and smooth, and
the surface roughness has to be substantially below the size
of objects under investigation. Substrate materials that
have been successfully used in SFM include: freshly cleaned
surfaces of highly orientated pyrolytic graphite (HOPG),
mica, evaporated or single crystal gold, glass slides and
silicon wafers, as known from the production of electronic
hardware. These surfaces can be further modified by chemical
reactions, in order to change their hydrophobicity, charge
and charge density, surface ion concentration and other parameters.
It is important, that the interaction between the sample
and the substrate surface is not too strong. Otherwise, the
morphology of the sample might be changed dramatically during
sample preparation. On the other hand, the interaction has
to be strong enough to prevent dislocation of the sample
during the scanning process. The size of the sample is limited
to 10 x 10 cm in x-y direction and about 1 cm in height.
Various methods are available for sample preparation. A very
convenient procedure involves the evaporation of a droplet
of the sample that has been dissolved or suspended in buffer.
A few micro litres are simply pipetted onto the substrate
which is then dried in the air. It is also possible to place
the sample as a dry power onto the substrate, but in this
case, the sample has to be homogeneous distributed over the
surface. In general, no fixation or staining of the sample
is needed.
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