direct investigation of individual vehiclesat high resolution in a hydrated state. Forinstance, there are various particle-sizingtechniques that produce a sizedistribution over a very large number ofcondensates, but they cannot be appliedto an individual condensate. One of themost common techniques currently used iselectron microscopy (EM), which offersthe high resolution needed to viewindividual condensates, but requiressignificant sample preparation time andrequires the specimen to be dried out ina vacuum environment. Since, drying outthe gene delivery vehicles may changetheir structure, the results are, at best, less useful.

DNA condensates that were formed with different charge relationships.Variables in the formation process result in either positive or negative chargedcondensates, producing varying degreesof DNA packing.

AFM offers a distinct advantage overother methods for investigating the DNAcondensates. One of the greatestadvantages is AFM’s ability to view thestructure of the delivery vehicle in itshydrated state, as it would appear in use.In addition, with AFM’s nanometer-scaleresolution, researchers can easily imagethe DNA strands and see how they reactand condense with a particular polymeror liposome. No other technique allows

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 with0.2 mM NiCl2, c) Condensed positively charged, d) Noncondensed. Depending on the formationmechanism, 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.

By maintaining a constant deflectionduring scanning, a constant vertical forceis maintained between the tip andsample. Although contact mode hasproven useful for a wide range ofapplications, it is not as effective onrelatively soft samples. On the otherhand, TappingMode AFM consists ofoscillating the cantilever at its resonancefrequency (typically ~300kHz) andscanning across the surface with aconstant, damped amplitude. Thefeedback loop maintains a constant root-mean-square (RMS) amplitude by movingthe scanner vertically during scanning,which correspondingly maintains aconstant applied force to form atopographic image. The advantage ofTappingMode is that it typically operateswith a lower vertical force than thatpossible with contact mode, and iteliminates 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 gainingmomentum as an effective method fortreating genetic-based diseases.However, one of the primary obstaclesfacing this form of treatment is in thedelivery of the condensed geneticmaterial to its intended target.6 There aretwo common methods of packing DNAfor gene delivery: viral and nonviral.Though the viral-mediated mode of genedelivery is currently the most common, itcan be problematic due to the activationof a patient’s immunological response,which may eliminate the gene deliveryvehicle before use and produce otherhealth problems. To avoid thesecomplications, nonviral vehiclesfabricated out of liposomes or polymersare increasingly being used toencapsulate the DNA or other drug-related materials. AFM has successfullyimproved the development of such genedelivery vehicles by providing a greaterunderstanding of the process of DNAcondensation. Figure 3 shows nonviral

a. b.

c. d.

Gene Delivery Condensation Studies

There have been a number of publishedAFM-based gene delivery researchstudies.6–14 Dunlap and coworkers studiedvariations in the condensationmechanisms of supercoiled plasmid DNA5–7kb in length with a cationic lipid,lipospermine (dioctadecylamido-glycylspermine or DOGS), and acationic polymer, polyethylenimine (PEI).7

The resulting condensates were imagedby TappingMode in 15mM NaClsolution. For both DOGS and PEI, foldedDNA loops radiating from central coreswere evident, indicating condensation bypacking folded loops of DNA. In Figure4, bundles of DNA can be clearly seenalong with the polymer globules. Thispartial condensate has formed a toroidalstructure from the circularization of anoblong condensate with multiplecondensation nodes. By varying theconcentration of DOGS and PEI, theconditions for complete condensationwere also investigated. Complete PEIcondensates were found to be 20 to 40 nanometers in diameter, whereascomplete DOGS condensates werefound to be 50 to 70 nanometers. Thissuggests that PEI may be a more effectivecondensing agent than the DOGS. Thedifference in size and morphology mayalso affect their efficiency as atransfection agent.

AFM has also been used to imagedynamic processes in solution to gain abetter understanding of the formationmechanisms associated with DNAcondensation.6 Condensation ofpegylated poly(amidoamine) with DNAhas been observed in aqueous solutionin real-time. The overall positive chargeof the polymer-DNA condensate wasused to electrostatically hold thecondensates to the negatively chargedmica. However, it was not held sorigidly as to prevent movement of thecondensates during their formation.Imaging of the condensates in solutionindicated the presence of toroidal and rod-like condensate structures. InFigure 5, the formation of a toroidalcondensate can be seen over a time-span 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 dynamicequilibrium, with the condensateschanging from rod-like to toroidal, andtoroidal to rod-like structures. Studyingthe dynamic processes in real-time makesit possible to gain a better understandingof the kinetics of the condensationprocess, which could lead to significantimprovements in the packaging ofgenetic material.

Generally, AFM is applied to genedelivery vehicles that are in a solution.They are injected into the fluid cantileverholder where they attach to a substrate,typically mica. The vehicles are held onthe mica surface by charge. Positivelycharged condensates are naturallyattracted to the negative charge of themica surface, and negatively chargedcondensates can be attracted to thenegatively charged mica by eitherplacing 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 straight-forward to prepare and perform, AFM isthus able to provide very high-resolutionon individual DNA condensates.

Figure 4. Nonviral gene delivery withcondensed DNA and a polymer. Orange-violet globules of polyethylenimine (PEI), acationic polymer, stabilized circular bundlesof yellow-green DNA loops in a five kilobaseplasmid. Unfixed molecules were imaged in TappingMode in 15mM salt solution.7

292nm x 292nm x 5nm scan courtesy D. Dunlap and A. Maggi, San RaffaeleScientific Institute, Italy.

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.