By maintaining a constant deflection
during scanning, a constant vertical force
is maintained between the tip andsample. Although contact mode has
proven useful for a wide range ofapplications, it is not as effective on
relatively soft samples. On the otherhand, TappingMode AFM consists ofoscillating the cantilever at its resonancefrequency (typically ~300kHz) andscanning across the surface with aconstant, damped amplitude. The
feedback loop maintains a constant rootmean-square (RMS) amplitude by moving
the scanner vertically during scanning,which correspondingly maintains aconstant applied force to form a
topographic image. The advantage ofTappingMode is that it typically operates
with a lower vertical force than thatpossible with contact mode, and it
eliminates the lateral, shear forces thatcan damage some samples. Thus,
TappingMode has become the preferredtechnique for imaging soft, fragile,adhesive, and particulate surfaces.

AFM in Gene Delivery Research
Gene therapy has been gaining
momentum as an effective method fortreating genetic-based diseases.
However, one of the primary obstacles
facing this form of treatment is in thedelivery of the condensed genetic
material to its intended target.6 There are
two common methods of packing DNA
for gene delivery: viral and nonviral.
Though the viral-mediated mode of genedelivery is currently the most common, it
can be problematic due to the activationof a patient’s immunological response,
which may eliminate the gene delivery
vehicle before use and produce other
health problems. To avoid thesecomplications, nonviral vehicles
fabricated out of liposomes or polymers
are increasingly being used toencapsulate the DNA or other drugrelated materials. AFM has successfully
improved the development of such genedelivery vehicles by providing a greater
understanding of the process of DNA
condensation. Figure 3 shows nonviral

a.

b.

c.

d.

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 NiCl 2, c) Condensed positively charged, d) Noncondensed. Depending on the formation
mechanism, the condensates are tightly packed or slightly unraveled. TappingMode AFM images inair. Images by Sophia Hohlbauch, Veeco Metrology Group; Samples courtesy of V. Trubetskoy,Mirus Corporation. 1µm scans.

DNA condensates that were formed
with different charge relationships.
Variables in the formation process resultin either positive or negative charged
condensates, producing varying degreesof DNA packing.
AFM offers a distinct advantage overother methods for investigating the DNAcondensates. One of the greatest
advantages is AFM’s ability to view thestructure 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 reactand 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 ofcondensates, but they cannot be applied
to an individual condensate. One of themost common techniques currently used iselectron microscopy (EM), which offers
the high resolution needed to view
individual condensates, but requires
significant sample preparation time andrequires the specimen to be dried out ina vacuum environment. Since, drying out
the gene delivery vehicles may change
their structure, the results are, at best,less useful.

Generally, AFM is applied to genedelivery vehicles that are in a solution.
They are injected into the fluid cantilever
holder 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 themica surface, and negatively charged
condensates can be attracted to thenegatively 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 thenconducted in solution via theTappingMode technique. Though straightforward to prepare and perform, AFM isthus able to provide very high-resolution
on individual DNA condensates.

Figure 5. Formation of toroidal DNA-polymer condensate over 35 minute time-span.6
Scale bar = 200nm. Images courtesy C. Roberts, University of Nottingham, UK.

Gene Delivery Condensation Studies

Figure 4. Nonviral gene delivery withcondensed DNA and a polymer. Orangeviolet globules of polyethylenimine (PEI), acationic polymer, stabilized circular bundlesof yellow-green DNA loops in a five kilobase
plasmid. Unfixed molecules were imaged
in TappingMode in 15mM salt solution. 7292nm x 292nm x 5nm scan courtesy
D. Dunlap and A. Maggi, San Raffaele
Scientific Institute, Italy.

There have been a number of published
AFM-based gene delivery researchstudies.6–14 Dunlap and coworkers studiedvariations in the condensation
mechanisms of supercoiled plasmid DNA
5–7kb in length with a cationic lipid,lipospermine (dioctadecylamidoglycylspermine or DOGS), and acationic polymer, polyethylenimine (PEI).7The resulting condensates were imaged
by TappingMode in 15mM NaClsolution. For both DOGS and PEI, folded
DNA loops radiating from central cores
were evident, indicating condensation bypacking folded loops of DNA. In Figure
4, bundles of DNA can be clearly seenalong with the polymer globules. Thispartial condensate has formed a toroidal
structure from the circularization of anoblong condensate with multiple
condensation nodes. By varying theconcentration of DOGS and PEI, theconditions for complete condensation
were also investigated. Complete PEI
condensates were found to be 20 to40 nanometers in diameter, whereas
complete DOGS condensates were
found to be 50 to 70 nanometers. Thissuggests that PEI may be a more effective
condensing agent than the DOGS. The
difference in size and morphology may
also affect their efficiency as atransfection agent.

AFM has also been used to image
dynamic processes in solution to gain abetter understanding of the formation
mechanisms associated with DNA
condensation.6 Condensation ofpegylated poly(amidoamine) with DNA
has been observed in aqueous solution
in real-time. The overall positive chargeof the polymer-DNA condensate wasused to electrostatically hold thecondensates to the negatively charged
mica. However, it was not held so
rigidly as to prevent movement of thecondensates during their formation.
Imaging of the condensates in solution
indicated the presence of toroidal
and rod-like condensate structures. InFigure 5, the formation of a toroidalcondensate can be seen over a timespan of 35 minutes. From these images,
the toroidal structure appears to beformed by joining the ends of a rod-likecondensate. Further investigations haveshown the existence of rod-like andtoroidal structures in a state of dynamic
equilibrium, with the condensateschanging from rod-like to toroidal, andtoroidal to rod-like structures. Studying
the dynamic processes in real-time makes
it possible to gain a better understandingof the kinetics of the condensation
process, which could lead to significant
improvements in the packaging ofgenetic material.