|
Microscopic evaluation is important for the
design and evaluation of a pharmaceutical product after the steps in the drug
formulation process. Since atomic force microscopy (AFM) provides the ability to
directly investigate surface structure at nanometer-to-subangstrom resolution in
ambient and liquid environments, it has been applied to a wide range of
pharmaceutical research, and delivers a powerful complement to other common
analytical techniques.
This application note describes the use of
AFM from the studies of drug crystal growth, particle characterization, and
tablet coatings in the manufacture of solid dosage forms.
AFM
Methods Used For Pharmaceutical Research
AFM is performed by scanning a sharp tip on
the end of a flexible cantilever across a sample surface, while maintaining a
small, constant force. Tip types vary depending on application requirements, but
they typically have an end radius of 5 to 10 nanometers. In a basic AFM setup, a
piezoelectric tube scanner scans the tip in a raster pattern over the sample.
Tip-sample interaction is monitored by reflecting a laser off the back of the
cantilever onto a split-photodiode detector.
Over the past several decades, a variety of
scanning modes have been developed to control how the tip scans the sample.
Contact mode and TappingMode™ are two of the more commonly used AFM techniques
of operation.
Contact
Mode
In contact mode AFM, a constant cantilever
deflection is maintained by a feedback loop that moves the scanner vertically
(Z) at each lateral (X,Y) data point to form the topographic image. By
maintaining a constant deflection during scanning, a constant vertical force is
maintained between the tip and sample. Applied forces during imaging typically
range between 0.1 and 100 nanonewtons. Although contact mode has proven useful
for a wide range of applications, it sometimes has difficulty on relatively soft
samples.
TappingMode
TappingMode AFM consists of oscillating the
cantilever at its resonance frequency (typically about 300 kilohertz) and
scanning across the surface with a constant, damped amplitude. The feedback loop
maintains a constant root-mean-square (RMS) amplitude by moving the scanner
vertically during scanning, which correspondingly maintains a constant applied
force to form a topographic image. The main advantage of TappingMode is that it
typically operates with a lower vertical force than contact mode, and it
eliminates the lateral, shear forces that can damage some samples.
Imaging
Soft, Fragile, Adhesive and Particulate Surfaces
Thus, TappingMode has become the preferred
technique for imaging soft, fragile, adhesive, and particulate surfaces.
Although the initial use of AFM was to produce high-resolution topographic
images, a number of related techniques have been developed to study the physical
and material properties of sample surfaces, resulting in the field of scanning
probe microscopy (SPM). For example, PhaseImaging™ consists of mapping the phase
lag of the oscillating cantilever with respect to the drive signal during
TappingMode imaging. This produces a topographic image along with a phase map
that can differentiate areas based on viscoelasticity, adhesion, hydrophobicity,
and other properties.
Crystal
Growth
Therapeutic agents are commonly formed into
crystalline forms for drug delivery. Three-dimensional surface morphology and
crystal structure have a dramatic effect on the manufacture, ease of delivery,
bioavailability, dissolution rate, and efficacy of crystalline drug forms. To
tailor the growth process to fit desired behavior, growth parameters such as
temperature, pH, concentration, and additive levels need to be optimized. In
situ visualization of crystallization has been conducted by AFM to optimize the
growth conditions in producing a desired morphology, as well as to study growth
mechanisms and defect formation.
For example, researchers Yip and Ward have
used AFM to study in situ crystallization characteristics of several insulin
forms: bovine insulin, LysB28ProB29 insulin,
ultralente insulin, and insulin-protamine complexes. Due to the low forces
needed to image the insulin surface, they used TappingMode imaging directly in
the crystallization liquor. Figure 1 shows the epitaxial growth of a screw
dislocation that was observed over 11 hours on LysB28ProB29 insulin.
Figure 1. In situ TappingMode AFM imaging of the (001) plane of
LysB28ProB29 insulin
during crystal growth. The imaging area is centered around a screw dislocation
that completes one rotation over an 11 hour period. Images were acquired at 1 =
0, 2 = 3605, 3 = 7210, 4 = 10,815, 5 = 18,025, and 6 = 40,590 seconds. In 1–3, a
defect (marked 1) intentionally formed by the tip is repaired within 120
minutes. In 4–6, an insulin aggregate forms a void that is not incorporated into
the growing terraces. 5ìm scans. Images courtesy of C. Yip,
University of
Toronto.
The terraces measure approximately 30
angstroms in height, which is consistent with the c-axis spacing of the insulin
hexamer of rhombohedral LysB28ProB29 insulin.
Observing the crystallization behavior in situ made it possible to determine
growth rates and observe defect formation in real-time. Step advancement was
observed to occur at 2 x 10-6 micrometers
per second, which corresponds to the attachment of approximately five unit cells
(15 LysB28ProB29 hexamers)
per second.
Defects appeared to be caused by large
insulin aggregates that were unable to align properly with the crystal structure
due to poor mobility, thus forming dislocations and voids in the growing
terraces.
These observations were conducted to study
differences in growth characteristics between LysB28ProB29 insulin and
wild-type porcine/bovine insulin. LysB28ProB29 insulin
differs from wild-type porcine/bovine insulins due to a sequence inversion at
the C-terminus in the B-chain. This sequence inversion was designed to reduce
the insulin monomer association for better dissolution properties. However, the
sequence inversion also produced differences in crystallization behavior, as
observed by AFM. LysB28ProB29 insulin was
shown to have smaller attachment energies (_Gk), more rounded screw
dislocations, larger terrace widths, and more persistent vacancies in the (001)
plane. These differences can have significant influence on the crystal quality
and on its behavior as a therapeutic agent.
Polymorphism
The ability of a drug substance to form into
more than one crystalline form is called polymorphism. Different polymorphs
possess different physicochemical properties, which affect solubility,
dissolution, adsorption, melting point, and stability. Thus, polymorphic
characterization is an important parameter in maintaining high product quality
and reproducibility in the pharmaceutical industry.
In Yip and Ward’s study on insulin discussed
above, the polymorphic forms of insulin were identified by using the AFM to map
the crystal structure by imaging the molecular lattice spacings in all three
dimensions. At the University of
Nottingham,
Danesh and coworkers used the advanced PhaseImaging technique to identify and
map the distribution of polymorphs of the drug cimetidine.
Force-interaction studies (amplitude-phase
distance relationships) were then conducted to identify the polymorphs based on
differences in hydrophobicity. In figure 2, cimetidine polymorphs A and B are
not easily distinguished in the topographic image, but their distribution is
easily characterized in the phase image. The contrast in the phase images is
most likely due to the differences in hydrophobicity between the polymorphs,
which produces a difference in the tip-sample interaction due to variations in
capillary force. This contrast was investigated by conducting the experiment
with hydrophilic (plasmaetched) and hydrophobic (alkylsilane) functionalized
probes.
Figure 2. Distribution of polymorphic forms A and B of the drug cimetidine
conducted by PhaseImaging with hydrophilic probes. The phase image (right) shows
the distribution of the A (dark) and B (light) polymorphs that is not evident in
the topographic TappingMode image (left). The phase image contrast is due to
differences in hydrophobicity. 4ìm scans. Images courtesy of C. Roberts,
University of
Nottingham.
Particles
Production of solid dosage forms commonly
begins with the formation of the drug into particles, typically within the size
of 0.1 to 10 micrometers. Characterization of these particles can be important
before drug formulation since their morphology, size, and shape can provide
information about the manufacturing process. Particle size has also been shown
to influence dissolution rate, bioavailability, content uniformity, stability,
texture, flow characteristics, and sedimentation rates, and thus has a
significant effect on formulation and therapeutic efficiency.
There are many methods commonly used to
investigate particles, such as light-scattering techniques like dynamic light
scattering and laser diffraction. However, these techniques sample a large
number of particles to provide a distribution of particle size or
characteristics. There are often cases where studying the particles individually
becomes a key step in understanding a particle system. One common method for
directly studying individual particles is transmission electron microscopy
(TEM). However, often the sample preparation of small particles for TEM is
challenging and time consuming. AFM has successfully examined pharmaceutical
particles directly to correlate their morphology to the manufacturing process
and behavioral properties.
Figure 3 shows an example of using the AFM to characterize the drug particle morphology. Drug particles are traditionally
formed by milling a drug crystal to particle sizes less than 10 micrometers by
micronization or spray-drying techniques.
Figure 3. Height (left column) and phase (right column) images of paracetamol
formed into drug particles by micronization and SEDS. Figure 3-1: raw starting
material showing crystalline lamellae. Figure 3-2: micronized particle showing
rough, irregular structure. Figure 3-3: SEDS particles showing regular, smooth
structure with 0.9nm crystalline steps. Figure 3-4: Roughness of starting
material, micronized particle, and SEDS particle. Images courtesy of Patel,
Davies, and Roberts, Molecular Profiles,
UK; and
Palakodaty, Gilbert, York, Bradford Particle Design Ltd.,
UK.
However, problems can result from these
techniques due to batch-to-batch variations, residual solvent, and
statically-charged particles that can affect powder stability and flow. Another
method of particle formation called solution enhanced dispersion by
supercritical fluids (SEDS) overcomes many of these problems and provides more
control of the particle size, shape, and morphology. The top left AFM image in
figure 3-1 shows the starting raw material of paracetamol in which crystalline
terraces can be seen. Images of particles formed by micronization and SEDS are
shown in figures 3-2 and 3-3 respectively.
Micronized
Particles
The micronized particles vary in size and
are irregular with a significant amount of surface roughness, whereas, the SEDS
particles have a regular shape and a size of approximately 10 micrometers, and
show a reduction in roughness from the raw starting material (see the bottom
right image of figure 3-4). As shown through AFM, the smoother surfaces and
regular shapes produced by SEDS should reduce the batch-to-batch variations and
static charge problems encountered with the micronized materials, as well as
improve the flow properties of the particles.
Granules
Once the drug is in particulate form, it is
often formed into a granule by mixing the drug particles with binding agents,
diluents, and disintegrating agents. The wet granulation process consists of
adding a liquid binder or adhesive to the mixture, passing the wetted mass
through a screen sieve of the desired mesh size, and drying the granules. The
resulting granules are typically in the range of a few millimeters, and show
improvements in flow properties as well as chemical and physical stability with
respect to particles. AFM has been very successful in characterizing the
morphology and roughness of granules to correlate their surface structure to the
underlying physicochemical and mechanical processes during the manufacturing
process (see figure 4).
Figure 4. Surface morphology of a wet granule of caffeine, lactose, and
polymers. Roughness measurements provide information about the formation process
and physicochemical properties. Inset images —Amplitude (left) and Height
(right) image. 5ìm scan. Image courtesy of T. Li, K.R. Morris, and K. Park,
Purdue
University.
Coatings
There are many coatings that may be applied
to tablets to serve various purposes. Common uses of pharmaceutical coatings
consist of protecting the drug from air and humidity, providing a barrier to an
objectionable tasting or smelling drug, and controlling the dissolution
behavior. Sugar coatings are very commonly applied to tablets, as well as
polymer coatings, which are more durable, less bulky, and less time-consuming to
apply. The polymer coatings are often designed to rupture in the
gastrointestinal tract to avoid stomach irritation and to improve drug
adsorption. Coating granules and other drug substances are also key steps in the
design of controlled-release and microencapsulated dosage forms.
AFM has been used commonly to correlate the
surface of coatings and thin films to deposition parameters (such as
temperature, rate, composition, etc.) and performance.
Common applications of AFM to investigate
coatings consist of evaluating the surface morphology, roughness, surface area,
compositional distribution, hardness, and porosity. Changes of these properties
have also been studied with respect to aging and environment. Figure 5 shows a
tablet coating that functions as a membrane for controlled-release applications
in which pores were formed during the membrane leaching and drug release
process. The pore structure, roughness, and surface area of the coating can
easily be determined by the AFM.
Figure 5. Tablet coating showing complex pore structure. Surface structure,
roughness, and surface area can be easily characterized. 5ìm scan. Sample
courtesy of ALZA Corporation.
Summary
AFM provides pharmaceutical researchers and
manufacturers with a wide variety of techniques to evaluate the steps of the
drug formulation process. The examples in this article indicate that with
high-resolution imaging in air and fluid environments, AFM has found utility in
the study of dynamic processes, fabrication variables, component distribution,
and structure-function relationships. With its capability for TappingMode and
PhaseImaging techniques, AFM provides information that cannot be acquired by
other analytical techniques. Thus, AFM is finding increasing use in the
pharmaceutical industry, which will undoubtedly lead to more applications and
the adoptions of other SPM techniques. |