**Conflict of interest**

*Current and Future Aspects of Nanomedicine*

described in Section 4.3.

for 2.0 h.

**4.3 Loading experiments: AGE**

to prevent auto-quenching.

**5. Conclusions**

**Acknowledgements**

the Morrison Trust.

during illumination with visible light [43].

have even more favorable characteristics.

Bleomycin was obtained from Cayman Chemical Company (Ann Arbor, MI, USA) as a powder. The bleomycin was dissolved in the aqueous buffer indicated and diluted to the concentrations indicated before incubation with capsids and DNA. Tests of fluorescence emission vs. fluorescent molecule concentration were made by pipetting 5 μl of diluted fluorescent molecule onto the surface of a 0.7% agarose gel (LE agarose, Lonza) that had been cast in a plastic Petri dish in the electrophoresis buffer of Section 4.3. The gel was then photographed by use of the procedures

To test for fluorescent compound/nanoparticle association, fluorescent compounds were mixed with one of the following T3 nanoparticles: NLD capsid II, NHD capsid II, phage, DNA. First, a 12.5 μl amount of fluorescent compound in 0.1 M NaCl, 0.01 M sodium citrate, pH 4.0, 0.001 M MgCl2 (citrate buffer) was added to 4.5 μl of additional citrate buffer. Then, 8.0 μl of a nanoparticle sample was added and mixed (final pH, 4.1). This mixture was incubated at 45.0°C

To perform AGE, we added to this mixture 2.5 μl of the following solution: 60% sucrose (to increase the density for layering in sample wells) in the electrophoresis buffer below. This final mixture was layered in a well of a horizontal, submerged, 0.7% agarose gel (LE agarose, Lonza), cast in and submerged under the following electrophoresis buffer: 0.05 M Tris-acetate, pH 8.4, 0.001 M MgCl2. The temperature of the gel and buffer had been pre-adjusted to 10°C in an effort to seal NLD

AGE was performed at 1.0 V/cm for 18.0 h with the gel and buffer maintained at 10°C by circulation through a controlled-temperature water bath. After AGE, the gel was soaked in 25% methanol in electrophoresis buffer for 1.5 h at room temperature, to cause leakage of fluorescent compounds from NLD capsid II and, therefore,

Finally, the gel was photographed during illumination with a Model TM-36 ultraviolet transilluminator (Ultra Violet Products, Inc.). The camera used was a Canon Power Shot G2, 4.0 Megapixels. The following Tiffin emission filters were used as described in Section 2: Blue, 80A #290513; Green, 11Green 1—#287305; Yellow, Yellow 12—#282224; Orange, Orange 16—#289750. To detect capsid protein, the gel was subsequently stained with Coomassie blue and photographed

Obtaining an increase in the current tumor-specificity of anticancer drugs should be possible via use of a DDV that implements multiple, independent stages of specificity increase. T3 NLD capsid II is an example of a bio-nanoparticle that has undergone some of the needed DDV-bioengineering via mutation/selection in the environment. Other examples, not yet found, are assumed to exist and potentially

The authors acknowledge support from the San Antonio Area Foundation and

capsid II and, therefore, prevent leakage of fluorescent compounds.

**20**

The authors declare no conflict of interest.
