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Atomic force microscopy (AFM) provides the
ability to perform three-dimensional measurements of surface structures at
nanometer-to-subangstrom resolution in ambient and liquid environments. These
capabilities have led to ground-breaking life sciences advances in the
investigation of DNA, proteins, and cells. In particular, pharmaceutical
research involves a number of applications that are rapidly benefiting from AFM,
both as a standalone technique and as a powerful complement to the other common
analytical techniques currently available.
This application note examines how AFM offers the unique capability of direct, individual investigation of gene
delivery vehicles at high resolution in a hydrated state.
AFM History and
Methods
AFM is the most commonly used form of the
scanning probe microscopy (SPM) family of techniques. The origin of SPM began
with the development of the scanning tunneling microscope (STM) in 1982 by
researchers at IBM, Zurich.
The ability of the STM to resolve atomic
structure on a sample surface earned the inventors the Nobel Prize in 1986.
However, the STM can only be applied to conductive or semiconductive specimens.
To broaden this type of microscopy to the study of insulators, the atomic force
microscope was developed in collaboration between IBM and
Stanford
University in
1986.
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 (see Figure 1).
Figure 1. SEM image (300X magnification) of an integrated single crystal
silicon cantilever and tip with an end radius of 5nm to
10nm.
The tips typically have an end radius of 5nm
to 10nm, although this can vary depending on tip type. The scanning motion is
conducted by a piezoelectric tube scanner that scans the tip over the sample in
a raster pattern (see Figure 2).
Figure 2. Schematic of the major components of an atomic force microscope,
showing the feedback loop for TappingMode operation.
TappingMode
and Contact Mode AFM
The tip-sample interaction is monitored by
reflecting a laser off the back of the cantilever onto a split-photodiode
detector. The two most commonly used modes of operation are contact mode AFM and
TappingMode™ AFM, which can be conducted in both air and liquid
environments.
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.
Although contact mode has proven useful for a wide range of applications, it is
not as effective on relatively soft samples. On the other hand, TappingMode AFM consists of oscillating the cantilever at its resonance frequency (typically
~300kHz) and scanning across the surface with a constant, damped amplitude. The
feedback loop maintains a constant rootmean-square (RMS) amplitude by moving the
scanner vertically during scanning, which correspondingly maintains a constant
applied force to form a topographic image. The advantage of TappingMode is that
it typically operates with a lower vertical force than that possible with
contact mode, and it eliminates the lateral, shear forces that can damage some
samples. Thus, TappingMode has become the preferred technique for imaging soft,
fragile, adhesive, and particulate surfaces.
AFM in Gene
Delivery Research
Gene therapy has been gaining momentum as an
effective method for treating genetic-based diseases.
However, one of the primary obstacles facing
this form of treatment is in the delivery of the condensed genetic material to
its intended target. There are two common methods of packing DNA for gene
delivery: viral and nonviral.
Though the viral-mediated mode of gene
delivery is currently the most common, it can be problematic due to the
activation of a patient’s immunological response, which may eliminate the gene
delivery vehicle before use and produce other health problems. To avoid these
complications, nonviral vehicles fabricated out of liposomes or polymers are
increasingly being used to encapsulate the DNA or other drug related materials.
AFM has successfully improved the development of such gene delivery vehicles by
providing a greater understanding of the process of DNA condensation. Figure 3
shows nonviral DNA condensates that were formed with different charge
relationships.
Figure 3. Four different condensed states of DNA from a study of nonviral gene
delivery vehicles: a) Condensed negatively charged on NiCl2-treated mica, b) Condensed negatively charged with 0.2 mM
NiCl2, c) Condensed
positively charged, d) Noncondensed. Depending on the formation mechanism, the
condensates are tightly packed or slightly unraveled. 1ìm
scans.
Variables in the formation process result in
either positive or negative charged condensates, producing varying degrees of
DNA packing.
AFM offers a distinct advantage over other
methods for investigating the DNA condensates. One of the greatest advantages is
AFM’s ability to view the structure of the delivery vehicle in its hydrated
state, as it would appear in use. In addition, with AFM’s nanometer-scale
resolution, researchers can easily image the DNA strands and see how they react
and condense with a particular polymer or liposome. No other technique allows
direct investigation of individual vehicles at high resolution in a hydrated
state. For instance, there are various particle-sizing techniques that produce a
size distribution over a very large number of condensates, but they cannot be
applied to an individual condensate. One of the most common techniques currently
used is electron microscopy (EM), which offers the high resolution needed to
view individual condensates, but requires significant sample preparation time
and requires the specimen to be dried out in a vacuum environment. Since, drying
out the gene delivery vehicles may change their structure, the results are, at
best, less useful. At worst, these results may be misleading and result in
ineffective gene delivery vehicles.
Generally, AFM is applied to gene delivery
vehicles that are in a solution. They are injected into the fluid cell where
they attach to a substrate, typically mica. The vehicles are held on the mica
surface by charge. Positively charged condensates are naturally attracted to the
negative charge of the mica surface, and negatively charged condensates can be
attracted to the negatively charged mica by either placing a divalent cation in
the solution, or by coating the mica with a silane to form AP-mica. AFM imaging
is then conducted in solution via the TappingMode technique. Though simple to
prepare and perform, AFM is thus able to provide very high-resolution on
individual DNA condensates.
Gene Delivery
Studies
There have been a number of published
AFM-based gene delivery studies. Dunlap and coworkers studied variations in the
condensation mechanisms of supercoiled plasmid DNA 5–7kb in length with a
cationic lipid, lipospermine (dioctadecylamidoglycylspermine or DOGS), and a
cationic polymer, polyethylenimine (PEI). The resulting
condensates were imaged by TappingMode in 15mM NaCl solution.
For both DOGS and PEI, folded DNA loops
radiating from central cores were evident, indicating condensation by packing
folded loops of DNA. In Figure 4, bundles of DNA can be clearly seen along with
the polymer globules. This partial condensate has formed a toroidal structure
from the circularization of an oblong condensate with multiple condensation
nodes. By varying the concentration of DOGS and
PEI, the conditions
for complete condensation were also investigated. Complete
PEI condensates were
found to be 20 to 40 nanometers in diameter, whereas complete DOGS condensates
were found to be 50 to 70 nanometers. This suggests that
PEI may be a more
effective condensing agent than the DOGS. The difference in size and morphology
may also affect their efficiency as a transfection agent.
Figure 4. Nonviral gene delivery with condensed DNA and a polymer.
Orangeviolet globules of polyethylenimine (PEI), a cationic
polymer, stabilized circular bundles of yellow-green DNA loops in a five
kilobase plasmid. Unfixed molecules were imaged in TappingMode in 15mM salt
solution.
AFM has also been used to image dynamic
processes in situ to gain a better understanding of the formation mechanisms
associated with DNA condensation. Condensation of pegylated poly(amidoamine)
with DNA has been observed in aqueous solution in real-time. The overall
positive charge of the polymer-DNA condensate was used to electrostatically hold
the condensates to the negatively charged mica. However, it was not held so
rigidly as to prevent movement of the condensates during their
formation.
Imaging of the condensates in solution
indicated the presence of toroidal and rod-like condensate structures. In Figure
5, the formation of a toroidal condensate can be seen over a timespan of 35
minutes. From these images, the toroidal structure appears to be formed by
joining the ends of a rod-like condensate.
Figure 5. Formation of toroidal DNA-polymer condensate over 35 minute
time-span. Scale bar = 200nm.
Further investigations have shown the
existence of rod-like and toroidal structures in a state of dynamic equilibrium,
with the condensates changing from rod-like to toroidal, and toroidal to
rod-like structures. Studying the dynamic processes in real-time makes it
possible to gain a better understanding of the kinetics of the condensation
process, which could lead to significant improvements in gene
delivery.
Conclusion
AFM has demonstrated success in studying
nanoscale, in situ DNA structures. This has an obvious relevance to efforts to
develop more effective gene delivery vehicles. Researchers are utilizing the
many benefits of AFM—high resolution, simplified sample preparation, real-time
investigation, non-destructive imaging, and the ability to perform in liquid—to
investigate DNA condensation mechanisms and various gene-packaging materials.
Although the examples discussed above are just a sampling of the work that has
been conducted with atomic force microscopes in gene delivery studies, they
indicate how important the AFM is to the future of gene therapy. The unique
benefits of AFM will almost certainly play a crucial role in gene therapy
development. |