**2. Results**

## **2.1 Detection of test compounds: GelStar**

We tested the loading of two fluorescent compounds. The first was GelStar, a fluorescent nucleic acid stain typically used after AGE. In contrast, we incubated GelStar with our capsid and then performed AGE without further use of GelStar. The second compound was bleomycin, an anticancer drug [37, 38] that is also fluorescent [38]. Neither the manufacturer nor the vendor provided either the structure of GelStar or the concentration of commercial GelStar solutions. GelStar is sold in solution only.

The dominant fluorescence emission of nucleic acid-bound GelStar is in the green range. Apparently not previously documented is that the dominant fluorescence emission of free GelStar is in the orange range, at least when the GelStar is in an agarose gel. Ultraviolet light stimulated GelStar fluorescence emission vs. GelStar dilution is shown in **Figure 2**. Free GelStar, at several dilutions, had been pipetted in 5 μl amounts onto an agarose gel before ultraviolet light illumination and photography through an orange filter (spots labeled G in **Figure 2**). The effective volume in μl (dilution, multiplied by 5) of the stock GelStar solution is also indicated. In **Figure 2**, the color of GelStar spots is orange for all dilutions, as it also is found to be (not shown) with yellow and green emission filters. The orange color is real and is not produced by the emission filter because green Alexa 488 dye fluorescence retains its green color (spots labeled A in **Figure 2**). The number next to the Alexa 488 spots is the total amount (μg) of Alexa 488, also applied in 5 μl amounts.

DNA-bound GelStar had the expected green emission at all dilutions, when viewed through the same orange emission filter used for **Figure 2** (right side of the right panel of **Figure 3**). Green emission was also dominant when yellow and green emission filters were used for DNA-bound GelStar (not shown).

#### **2.2 Detection of test compounds: bleomycin**

Without fluorescent compound, the background of an agarose gel was blue when emission was photographed without an emission filter (not shown). Thus, not surprising was that optimal detection of bleomycin was not obtained with a blue filter, even though the blue range was where peak emission was previously found for bleomycin [38]. Among the blue, green, yellow and orange filters, optimal detection was obtained with the green filter.

#### **Figure 2.**

*Fluorescence of free GelStar. GelStar and Alexa Fluor 488 were diluted and, then, pipetted onto the surface of an agarose gel. The fluorescence was photographed through the orange filter. The GelStar samples are indicated by G, followed by the effective volume (μl) of the original, undiluted GelStar solution. The Alexa Fluor 488 samples are indicated by A, followed by the amount (μg) of Alexa Fluor 488.*

**17**

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles*

With the green emission filter, the minimal detected bleomycin amount was 0.2–0.4 ng when a bleomycin dilution series like the GelStar dilution series in **Figure 2** was photographed (not shown). Contrast enhancement of images was

*Association of GelStar with T3 NLD capsid II. A commercial GelStar solution was diluted to the extent indicated above a lane and incubated with T3 NLD capsid II. Association of GelStar with the capsid was determined by AGE, followed by, first, photography of fluorescence (right panel, lanes labeled NLD CII) and, then, staining of protein with Coomassie blue (left panel). Also analyzed was purified T3 DNA (right panel, lanes labeled DNA), which does not stain with Coomassie blue. The arrow indicates the direction of* 

We succeeded in loading GelStar into NLD capsid II. To achieve loading, 10 μg of NLD capsid II was incubated with GelStar at 45°C. Loading was then assayed by AGE at 10°C. Then, the gel was illuminated with ultraviolet light. The result was a fluorescent band of intensity that monotonically increased with decreasing GelStar dilution (left section of the right panel of **Figure 3**). The capsid amount was invariant, as judged by Coomassie staining of the same gel (left panel of **Figure 3**). At GelStar dilutions lower than those in **Figure 3**, down to 1/10, the band intensity reached a plateau (not shown). The dominant fluorescence, at all dilutions, was green, implying that the GelStar was bound to something capsid associated. GelStar

The following data indicated that the GelStar-binding capsid site was not on a DNA molecule associated with the capsid. As the dilution of GelStar decreased, the DNA-bound GelStar fluorescence underwent, first, an increase and then a decrease (**Figure 3**, right segment of right panel). However, the decrease was not observed for the binding to NLD capsid II. Second, although a minor NLD capsid II fraction has DNA [31], the DNA-containing NLD capsid II had been excluded during purification by selecting the low-density side of the NLD capsid II band after buoyant density centrifugation in a Nycodenz density gradient. Thus, the GelStar

Association of bleomycin with T3 NLD capsid II was also achieved. However, the fluorescence signal was relatively weak (**Figure 4**). The bleomycin fluorescence signal of a NLD capsid II band did not change when the concentration of bleomycin was changed from 2 to 16 mg/ml. A bleomycin-associated NLD capsid II band is

*DOI: http://dx.doi.org/10.5772/intechopen.91052*

used at these lower amounts.

**Figure 3.**

**2.3 Loading of GelStar in NLD capsid II**

*electrophoresis; the arrowheads indicate the origins.*

did not detectably associate with NHD capsid II (not shown).

was apparently either self-bound or bound to capsid protein.

**2.4 Loading of bleomycin in NLD capsid II**

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles DOI: http://dx.doi.org/10.5772/intechopen.91052*

**Figure 3.**

*Current and Future Aspects of Nanomedicine*

**2.1 Detection of test compounds: GelStar**

We tested the loading of two fluorescent compounds. The first was GelStar, a fluorescent nucleic acid stain typically used after AGE. In contrast, we incubated GelStar with our capsid and then performed AGE without further use of GelStar. The second compound was bleomycin, an anticancer drug [37, 38] that is also fluorescent [38]. Neither the manufacturer nor the vendor provided either the structure of GelStar or the concentration of commercial GelStar solutions. GelStar is sold in solution only. The dominant fluorescence emission of nucleic acid-bound GelStar is in the green range. Apparently not previously documented is that the dominant fluorescence emission of free GelStar is in the orange range, at least when the GelStar is in an agarose gel. Ultraviolet light stimulated GelStar fluorescence emission vs. GelStar dilution is shown in **Figure 2**. Free GelStar, at several dilutions, had been pipetted in 5 μl amounts onto an agarose gel before ultraviolet light illumination and photography through an orange filter (spots labeled G in **Figure 2**). The effective volume in μl (dilution, multiplied by 5) of the stock GelStar solution is also indicated. In **Figure 2**, the color of GelStar spots is orange for all dilutions, as it also is found to be (not shown) with yellow and green emission filters. The orange color is real and is not produced by the emission filter because green Alexa 488 dye fluorescence retains its green color (spots labeled A in **Figure 2**). The number next to the Alexa 488 spots is the total amount (μg) of Alexa 488, also applied in 5 μl amounts. DNA-bound GelStar had the expected green emission at all dilutions, when viewed through the same orange emission filter used for **Figure 2** (right side of the right panel of **Figure 3**). Green emission was also dominant when yellow and green

emission filters were used for DNA-bound GelStar (not shown).

Without fluorescent compound, the background of an agarose gel was blue when emission was photographed without an emission filter (not shown). Thus, not surprising was that optimal detection of bleomycin was not obtained with a blue filter, even though the blue range was where peak emission was previously found for bleomycin [38]. Among the blue, green, yellow and orange filters, optimal

*Fluorescence of free GelStar. GelStar and Alexa Fluor 488 were diluted and, then, pipetted onto the surface of an agarose gel. The fluorescence was photographed through the orange filter. The GelStar samples are indicated by G, followed by the effective volume (μl) of the original, undiluted GelStar solution. The Alexa Fluor 488* 

*samples are indicated by A, followed by the amount (μg) of Alexa Fluor 488.*

**2.2 Detection of test compounds: bleomycin**

detection was obtained with the green filter.

**2. Results**

**16**

**Figure 2.**

*Association of GelStar with T3 NLD capsid II. A commercial GelStar solution was diluted to the extent indicated above a lane and incubated with T3 NLD capsid II. Association of GelStar with the capsid was determined by AGE, followed by, first, photography of fluorescence (right panel, lanes labeled NLD CII) and, then, staining of protein with Coomassie blue (left panel). Also analyzed was purified T3 DNA (right panel, lanes labeled DNA), which does not stain with Coomassie blue. The arrow indicates the direction of electrophoresis; the arrowheads indicate the origins.*

With the green emission filter, the minimal detected bleomycin amount was 0.2–0.4 ng when a bleomycin dilution series like the GelStar dilution series in **Figure 2** was photographed (not shown). Contrast enhancement of images was used at these lower amounts.

#### **2.3 Loading of GelStar in NLD capsid II**

We succeeded in loading GelStar into NLD capsid II. To achieve loading, 10 μg of NLD capsid II was incubated with GelStar at 45°C. Loading was then assayed by AGE at 10°C. Then, the gel was illuminated with ultraviolet light. The result was a fluorescent band of intensity that monotonically increased with decreasing GelStar dilution (left section of the right panel of **Figure 3**). The capsid amount was invariant, as judged by Coomassie staining of the same gel (left panel of **Figure 3**). At GelStar dilutions lower than those in **Figure 3**, down to 1/10, the band intensity reached a plateau (not shown). The dominant fluorescence, at all dilutions, was green, implying that the GelStar was bound to something capsid associated. GelStar did not detectably associate with NHD capsid II (not shown).

The following data indicated that the GelStar-binding capsid site was not on a DNA molecule associated with the capsid. As the dilution of GelStar decreased, the DNA-bound GelStar fluorescence underwent, first, an increase and then a decrease (**Figure 3**, right segment of right panel). However, the decrease was not observed for the binding to NLD capsid II. Second, although a minor NLD capsid II fraction has DNA [31], the DNA-containing NLD capsid II had been excluded during purification by selecting the low-density side of the NLD capsid II band after buoyant density centrifugation in a Nycodenz density gradient. Thus, the GelStar was apparently either self-bound or bound to capsid protein.

#### **2.4 Loading of bleomycin in NLD capsid II**

Association of bleomycin with T3 NLD capsid II was also achieved. However, the fluorescence signal was relatively weak (**Figure 4**). The bleomycin fluorescence signal of a NLD capsid II band did not change when the concentration of bleomycin was changed from 2 to 16 mg/ml. A bleomycin-associated NLD capsid II band is

#### **Figure 4.**

*Association of bleomycin with T3 NLD capsid II. The experiment of Figure 3 was repeated with bleomycin (8 mg/ml), instead of GelStar. The capsid region of the post-AGE gel is shown. The right (protein) panel has a single band of capsid stained with Coomassie blue. This band marks the position of the capsid-associated bleomycin fluorescence in the left panel. The arrow indicates direction of electrophoresis.*

shown in **Figure 4**. Most of the free bleomycin migrated toward the cathode (not shown), i.e., in a direction opposite to the direction of capsid migration.

The strength of the signal in **Figure 4** was weakened by the blue background and use of a green filter. In addition, a contaminant in the bleomycin preparation migrated close to the capsids, and is seen above the capsid band at the top of the left panel of **Figure 4**.

Calibration data for bleomycin, like the data for GelStar in **Figure 2**, were obtained. These data revealed that the amount of bleomycin loaded was 150–300 molecules per capsid.

### **3. Discussion**

In the Introduction, we outlined a strategy that is expected to work, if we can achieve the following objectives: (1) high (~4 h) persistence of NLD capsid II in blood so that the EPR effect has time to work, (2) adequate loading and sealing of NLD capsid II and (3) tumor-specific, controlled release (de-sealing or unloading). Objective #1 is likely already achieved, given the high persistence of T3 phage. That is to say, if one considers this strategy to be engineering based, some of the engineering might already be been done by natural selection.

Concerning adequate loading, the volume of the internal cavity of NLD capsid II = 6.95 × 10<sup>−</sup>17 ml. For volume occupancy (*F*V) of 0.5 (equal to the *F*V of DNA packaged in mature phage [34]), the number of bleomycin molecules (1416 Da; density estimated at 1.6 g/ml as sulfate) per NLD capsid II particle is 2.4 × 104 . The recommended dose of DDV-free bleomycin depends on the tumor, but is typically [39, 40] 10–20 units/m<sup>2</sup> , corresponding roughly to 10–20 mg/m<sup>2</sup> ; 15 mg/m2 is 1.76 × 1018 bleomycin molecules/m2 .

To calculate the number (*N*D) of NLD capsid II particles needed for this dose at *F*V = 0.5, we initially assume a 25 g mouse, which on average, has 78.6 cm<sup>2</sup> surface area [41]. Then, *N*D is 3.6 × 1011. A 6-liter culture yields 150–300 mouse doses of this size (cost ~ \$1500), assuming (1) laboratory-scale production technique, (2) no development of procedures to increase the amount produced per bacterial cell, and (3) no drug-dose reduction caused by improved targeting. That is to say, if we can half-fill the volume of NLD capsid II, we have a viable beginning. However, thus far, we have filled no more than 2% of *F*V = 0.5, NLD capsid II volume. So, increasing the loading is a major objective for the future.

An apparent obstacle to achieving this goal is the nonincrease in loading as bleomycin concentration increases above 2 mg/ml. At least two possible explanations

**19**

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles*

exist. (1) After passing through an open gate, the bleomycin eventually causes the gate to close. We were hoping to close the gate by lowering temperature. (2) After diffusing through an open gate, the bleomycin is prevented from diffusing in reverse by binding to internal proteins; the internal proteins become saturated as the concentration of bleomycin increases. In either case, increasing the loading is a

An advantage of using a phage DDV is that the human-design engineering potential is relatively high. First of all, the capsids in a T7 NLD capsid II preparation are structurally uniform enough so that symmetric cryo-EM reconstruction is obtained at 3.5 Å [34] and asymmetric reconstruction, at ~8 Å [36]. Assuming T3 capsids to be comparably homogeneous, use of chemistry to improve gating should

Second, phages, in general, and phage T3 in particular, can be genetically manipulated, which is not possible with liposomes. Information for determining which nucleotides to change can be obtained from high-resolution cryo-EM struc-

of an NLD capsid II-like capsid are the related coliphages, T7, T3 and ϕII. All three of these phages produce a NLD capsid II-like capsid [42]. Other phages are potential sources of gated capsids, perhaps with properties even more

We obtained bacteriophage T3 and T3 capsid II from 30°C-lysates of host, *Escherichia coli* BB/1, that had been infected by phage T3 in aerated liquid culture [43]. The growth medium was 2× LB medium: 2.0% Bacto tryptone, 1.0% Bacto yeast in 0.1 M NaCl. We initially purified both phage and capsids by centrifugation through a cesium chloride step gradient, followed by buoyant density centrifugation in a cesium chloride density gradient [43]. The latter fractionation separates

To separate NLD capsid II from NHD capsid II, we performed buoyant density centrifugation of capsid II in a Nycodenz density gradient, as previously described [32]. The purified NLD and NHD capsid II were dialyzed against 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M MgCl2. NLD capsid II, which formed a band near the top of the Nycodenz density gradient, had no detected contamination with NHD capsid II and vice versa, as previously seen by analytical ultracentrifugation [31]. Phage, NLD capsid II and NHD capsid II were dialyzed against the following buffer before use in the experiments described below: 0.2 M NaCl, 0.01 M Tris-Cl,

T3 DNA was obtained from purified T3 phage by phenol extraction. The DNA was dialyzed against and stored in 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M EDTA. DNA concentration was obtained from optical density at 260 nm.

GelStar was obtained from Lonza (Basel, Switzerland) in solution. The company recommends dilution by a factor of 1:10,000 for use as a nucleic acid stain after gel electrophoresis. Alexa Fluor 488 succinimidyl ester was obtained from Molecular

Finally, we note that, as far as we know, the only phages tested for production

ture. Structure of this type is not obtainable with liposomes.

**4.1 T3 bacteriophage, capsids and DNA (nanoparticles)**

**4.2 Fluorescent compounds: test of fluorescence emission**

Probes (Eugene, OR, USA) as a powder.

*DOI: http://dx.doi.org/10.5772/intechopen.91052*

problem of engineering.

DDV-favorable.

**4. Materials and methods**

capsid I from capsid II.

pH 7.4, 0.001 MgCl2.

produce relatively uniform results.

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles DOI: http://dx.doi.org/10.5772/intechopen.91052*

exist. (1) After passing through an open gate, the bleomycin eventually causes the gate to close. We were hoping to close the gate by lowering temperature. (2) After diffusing through an open gate, the bleomycin is prevented from diffusing in reverse by binding to internal proteins; the internal proteins become saturated as the concentration of bleomycin increases. In either case, increasing the loading is a problem of engineering.

An advantage of using a phage DDV is that the human-design engineering potential is relatively high. First of all, the capsids in a T7 NLD capsid II preparation are structurally uniform enough so that symmetric cryo-EM reconstruction is obtained at 3.5 Å [34] and asymmetric reconstruction, at ~8 Å [36]. Assuming T3 capsids to be comparably homogeneous, use of chemistry to improve gating should produce relatively uniform results.

Second, phages, in general, and phage T3 in particular, can be genetically manipulated, which is not possible with liposomes. Information for determining which nucleotides to change can be obtained from high-resolution cryo-EM structure. Structure of this type is not obtainable with liposomes.

Finally, we note that, as far as we know, the only phages tested for production of an NLD capsid II-like capsid are the related coliphages, T7, T3 and ϕII. All three of these phages produce a NLD capsid II-like capsid [42]. Other phages are potential sources of gated capsids, perhaps with properties even more DDV-favorable.

### **4. Materials and methods**

*Current and Future Aspects of Nanomedicine*

panel of **Figure 4**.

**Figure 4.**

molecules per capsid.

[39, 40] 10–20 units/m<sup>2</sup>

1018 bleomycin molecules/m2

**3. Discussion**

shown in **Figure 4**. Most of the free bleomycin migrated toward the cathode (not

*Association of bleomycin with T3 NLD capsid II. The experiment of Figure 3 was repeated with bleomycin (8 mg/ml), instead of GelStar. The capsid region of the post-AGE gel is shown. The right (protein) panel has a single band of capsid stained with Coomassie blue. This band marks the position of the capsid-associated* 

Calibration data for bleomycin, like the data for GelStar in **Figure 2**, were obtained. These data revealed that the amount of bleomycin loaded was 150–300

In the Introduction, we outlined a strategy that is expected to work, if we can achieve the following objectives: (1) high (~4 h) persistence of NLD capsid II in blood so that the EPR effect has time to work, (2) adequate loading and sealing of NLD capsid II and (3) tumor-specific, controlled release (de-sealing or unloading). Objective #1 is likely already achieved, given the high persistence of T3 phage. That is to say, if one considers this strategy to be engineering based, some of the engi-

Concerning adequate loading, the volume of the internal cavity of NLD capsid II = 6.95 × 10<sup>−</sup>17 ml. For volume occupancy (*F*V) of 0.5 (equal to the *F*V of DNA packaged in mature phage [34]), the number of bleomycin molecules (1416 Da; density estimated at 1.6 g/ml as sulfate) per NLD capsid II particle is 2.4 × 104

recommended dose of DDV-free bleomycin depends on the tumor, but is typically

area [41]. Then, *N*D is 3.6 × 1011. A 6-liter culture yields 150–300 mouse doses of this size (cost ~ \$1500), assuming (1) laboratory-scale production technique, (2) no development of procedures to increase the amount produced per bacterial cell, and (3) no drug-dose reduction caused by improved targeting. That is to say, if we can half-fill the volume of NLD capsid II, we have a viable beginning. However, thus far, we have filled no more than 2% of *F*V = 0.5, NLD capsid II volume. So, increasing

*F*V = 0.5, we initially assume a 25 g mouse, which on average, has 78.6 cm2

, corresponding roughly to 10–20 mg/m<sup>2</sup>

To calculate the number (*N*D) of NLD capsid II particles needed for this dose at

An apparent obstacle to achieving this goal is the nonincrease in loading as bleomycin concentration increases above 2 mg/ml. At least two possible explanations

. The

is 1.76 ×

surface

; 15 mg/m2

neering might already be been done by natural selection.

.

the loading is a major objective for the future.

The strength of the signal in **Figure 4** was weakened by the blue background and use of a green filter. In addition, a contaminant in the bleomycin preparation migrated close to the capsids, and is seen above the capsid band at the top of the left

shown), i.e., in a direction opposite to the direction of capsid migration.

*bleomycin fluorescence in the left panel. The arrow indicates direction of electrophoresis.*

**18**

#### **4.1 T3 bacteriophage, capsids and DNA (nanoparticles)**

We obtained bacteriophage T3 and T3 capsid II from 30°C-lysates of host, *Escherichia coli* BB/1, that had been infected by phage T3 in aerated liquid culture [43]. The growth medium was 2× LB medium: 2.0% Bacto tryptone, 1.0% Bacto yeast in 0.1 M NaCl. We initially purified both phage and capsids by centrifugation through a cesium chloride step gradient, followed by buoyant density centrifugation in a cesium chloride density gradient [43]. The latter fractionation separates capsid I from capsid II.

To separate NLD capsid II from NHD capsid II, we performed buoyant density centrifugation of capsid II in a Nycodenz density gradient, as previously described [32]. The purified NLD and NHD capsid II were dialyzed against 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M MgCl2. NLD capsid II, which formed a band near the top of the Nycodenz density gradient, had no detected contamination with NHD capsid II and vice versa, as previously seen by analytical ultracentrifugation [31]. Phage, NLD capsid II and NHD capsid II were dialyzed against the following buffer before use in the experiments described below: 0.2 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 MgCl2.

T3 DNA was obtained from purified T3 phage by phenol extraction. The DNA was dialyzed against and stored in 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M EDTA. DNA concentration was obtained from optical density at 260 nm.

#### **4.2 Fluorescent compounds: test of fluorescence emission**

GelStar was obtained from Lonza (Basel, Switzerland) in solution. The company recommends dilution by a factor of 1:10,000 for use as a nucleic acid stain after gel electrophoresis. Alexa Fluor 488 succinimidyl ester was obtained from Molecular Probes (Eugene, OR, USA) as a powder.

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 described in Section 4.3.

#### **4.3 Loading experiments: AGE**

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 for 2.0 h.

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

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, to prevent auto-quenching.

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 during illumination with visible light [43].

#### **5. Conclusions**

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 have even more favorable characteristics.

#### **Acknowledgements**

The authors acknowledge support from the San Antonio Area Foundation and the Morrison Trust.

**21**

**Author details**

Philip Serwer\*, Elena T. Wright and Cara B. Gonzales

\*Address all correspondence to: serwer@uthscsa.edu

provided the original work is properly cited.

The University of Texas Health Science Center, San Antonio, Texas, USA

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles*

*DOI: http://dx.doi.org/10.5772/intechopen.91052*

The authors declare no conflict of interest.

**Conflict of interest**

*Phage Capsids as Gated, Long-Persistence, Uniform Drug Delivery Vehicles DOI: http://dx.doi.org/10.5772/intechopen.91052*
