**Abstract**

Over the last 25 years, cancer therapies have improved survivorship. Yet, metastatic cancers remain deadly. Therapies are limited by inadequate targeting. Our goal is to develop a new drug delivery vehicle (DDV)-based strategy that improves targeting of drug delivery to solid tumors. We begin with a capsid nanoparticle derived from bacteriophage (phage) T3, a phage that naturally has high persistence in murine blood. This capsid has gating capacity. For rapidly detecting loading in this capsid, here, we describe procedures of native agarose gel electrophoresis, coupled with fluorescence-based detection of loaded molecules. We observe the loading of two fluorescent compounds: the dye, GelStar, and the anticancer drug, bleomycin. The optimal emission filters were found to be orange and green, respectively. The results constitute a first milestone in developing a drug-loaded DDV that does not leak when in blood, but unloads its cargo when in a tumor.

**Keywords:** agarose gel electrophoresis, bacteriophage T3, bleomycin, buoyant density centrifugation, capsid impermeability, GelStar

## **1. Introduction**

Current therapies for cancerous tumors suffer from both toxic secondary effects and the development by the tumor of drug resistance. These effects usually block therapy for metastatic cancers, the cause of 90% of cancer deaths [1–5]. For solving these problems, our first thesis is that the best strategy is to increase tumor specificity of anticancer drug delivery in several, *independent* stages. If, for example, three stages are used and each stage is 80% efficient (20% nonefficient) in increasing specificity, then overall efficiency is 99% [100 × (1.0 − 0.2<sup>3</sup> )]. In this case, (1) drug dosages to tumors can be raised 100× without changing toxicity and, therefore, (2) tumor cell evolution of drug resistance is minimized.

The primary alternative is to continue testing chemotherapies [6–8], immunotherapies [9–11] and radiotherapies [12–14] that have tumor-specificity determined at one independent stage. This one stage is often cellular DNA replication, which is more rapid and, therefore, more drug- and radiation-sensitive, in cancerous cells than it is in healthy cells. One-stage strategies are >100 years old for immunotherapy and radiotherapy. Chemotherapeutic agents typically used are over 50 years old [8]. Even major effort has not produced systematic therapies for metastatic cancer.

Apparently, new, possibly more biology-based, strategy is needed to counter risk that the above one-stage-based strategies, in general, are not realistic for reaching objectives (reviewed in [1, 15–17]).

In theory, one implementation of multi-stage strategy starts with drug delivery in a drug delivery vehicle (DDV) that is gated. The gate is opened to load drug, closed in circulation to deliver drug and opened again in tumors to administer drug. Stages of DDV-derived toxicity reduction are the following tumor-specific events: (1) DDV delivery, (2) DDV opening and, (3) drug activation, for masked drugs. We address stages (1) and (2) here.

In this implementation, tumor-specific delivery is achieved via the EPR effect. The EPR effect is the spontaneous accumulation of nanoparticles in tumors, observed for an uncharacterized phage in 1940 [18] (reviewed in [19]). The causes of the EPR effect are (1) porosity of tumor blood vessels and relative tightness of healthy blood vessels, so that nanoparticles enter tumors, but usually not healthy tissue and (2) poor tumor lymphatic drainage, so that nanoparticles remain [20–25]. The EPR effect is the basis for the use of several FDA-approved, drugloaded, liposomal DDVs [23, 24, 26].

However, circulating, drug-loaded, liposomal DDVs undergo drug leakage that causes significant toxicity [22, 23, 25, 27]. Also, liposomes are removed from circulation by the macrophage-phagocyte innate immune system. That is to say, liposomes are not very persistent. Chemical solutions to the leakage problem do not exist to our knowledge. Chemical solutions to the persistence problem (e.g., polyethylene glycol derivatization [28, 29]) introduce quality control problems and are not adaptable to future improvements, for example, achieving of tumorspecific unloading. The second thesis is that the optimal solution is linked to finding an appropriate, biologically produced, microbial DDV. Unlike some biology-based anticancer strategies [1, 15, 16, 19], use of a DDV is implementable with nononcolytic viruses and, therefore, avoids the dangers [19] of using oncolytic viruses.

In practice, we have discovered a phage T3 capsid that appears to have DDVfavorable characteristics needed for implementation of our strategy. First, phage T3 (and presumably its capsid) has recently been found to have exceptionally high persistence in mouse blood (3–4 h), unlike the T3 relative, T7 [30]. Second, one empty, but otherwise phage-like T3 capsid, is impermeable (for over 20 years) to the compound, Nycodenz (821 Da molecular weight) (reviewed in [31]). But, when the temperature is raised to 45°C, Nycodenz enters this capsid [32], presumably through a gate that opened. The concept is that, if we adapt this capsid to use as gated DDV, some of the needed engineering has already been done by natural selection.

Phages T3 and T7 are illustrated at the left in **Figure 1**. The gated capsid is illustrated at the right in **Figure 1**. This capsid is generated during DNA packaging that had been initiated by a DNA-free procapsid called capsid I (not shown). During packaging, capsid I expands and becomes the more angular and stable capsid (capsid II) illustrated in **Figure 1**. In nature, the capsid-gate is a ring of 12 gp8 molecules (**Figure 1**) that acts as entry portal for DNA during DNA packaging [31–33]. Most T3 and T7 capsid II particles are purified after having detached from DNA during infected cell lysis. The amount is 5–10 mg capsid II per liter of culture. The last purification step is buoyant density centrifugation in a Nycodenz or Metrizamide density gradient. Nycodenz low density (NLD) capsid II has the gp8 "gate" and is impermeable to Nycodenz and Metrizamide. The low density (1.08 g/ml) is caused by high internal hydration, which is caused by Nycodenz impermeability.

**15**

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

Nycodenz high density (NHD) capsid II is Nycodenz-permeable (1.28 g/ml) and is completely separated from NLD capsid II during buoyant density centrifugation in a Nycodenz or Metrizamide density gradient [31, 33–35]. We use NHD capsid II as a

*Phages T3 and T7 (left); and T3 and T7 NLD capsid II (right). The graphic legend at the top indicates the various capsid components. A protein is labeled by gp, followed by the number of the encoding gene. Analogous T3 and T7 genes have the same numbers. If NLD capsid II is used as DDV, the perimeter of the DDV is defined by the gp10 shell; the gate is the gp8 portal. All structures are the same for phage T3 as they are for phage T7* 

In the case of T3 NLD capsid II, gated entry of Nycodenz has been observed via raising of temperature. The likely entry channel was the axial hole of the gp8 ring. This conclusion was drawn, in the case of T7 NLD capsid II, from (1) entry kinetics of the fluorescent dye, bis-ANS [1,1′-bi(4-anilino)naphthalene-5,5′-di-sulfonic acid; 673 Da], and (2) covalent cross-linking of bis-ANS to channel proteins [33]. Asymmetric reconstruction-cryo-EM [36] revealed that obstruction of the T7 gp8

In support of working to implement the above gating-based strategy, the following quote from 2005 presents an expert opinion of what is needed for the next generation of anticancer DDVs [27]. "An ideal liposomal anticancer drug would exhibit little or no drug release while in the plasma compartment, thus ensuring limited exposure of the drug to healthy tissue. This feature would also maximize drug delivery to disease sites, as mediated by the movement of the drug-loaded liposomes from the plasma compartment to the extravascular space at disease sites, such as a region of tumor growth. Following localization, however, the drug-loaded liposome must transform itself from a stable carrier to an unstable carrier. This would ensure that the drug, which has localized in the diseased site, is bioavailable." To our knowledge, no details for such "controlled release" via a liposomal DDV have been published. The system described here is designed to accomplish what is described in the above quote. However, implementation uses gating of a DDV, not

To proceed further, we need a procedure for rapidly determining whether a drug

is loaded in a capsid. In the current study, we have developed native agarose gel electrophoresis (AGE) for this purpose. The detection is performed via capsid band

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

control during loading experiments.

**Figure 1.**

*(reviewed in [31, 32]).*

programmed instability of a DDV.

fluorescence produced by the compound loaded.

channel (and presumably the T3 channel) varies.

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

#### **Figure 1.**

*Current and Future Aspects of Nanomedicine*

objectives (reviewed in [1, 15–17]).

address stages (1) and (2) here.

loaded, liposomal DDVs [23, 24, 26].

lytic viruses.

selection.

Apparently, new, possibly more biology-based, strategy is needed to counter risk that the above one-stage-based strategies, in general, are not realistic for reaching

In theory, one implementation of multi-stage strategy starts with drug delivery in a drug delivery vehicle (DDV) that is gated. The gate is opened to load drug, closed in circulation to deliver drug and opened again in tumors to administer drug. Stages of DDV-derived toxicity reduction are the following tumor-specific events: (1) DDV delivery, (2) DDV opening and, (3) drug activation, for masked drugs. We

In this implementation, tumor-specific delivery is achieved via the EPR effect.

However, circulating, drug-loaded, liposomal DDVs undergo drug leakage that causes significant toxicity [22, 23, 25, 27]. Also, liposomes are removed from circulation by the macrophage-phagocyte innate immune system. That is to say, liposomes are not very persistent. Chemical solutions to the leakage problem do not exist to our knowledge. Chemical solutions to the persistence problem (e.g., polyethylene glycol derivatization [28, 29]) introduce quality control problems and are not adaptable to future improvements, for example, achieving of tumorspecific unloading. The second thesis is that the optimal solution is linked to finding an appropriate, biologically produced, microbial DDV. Unlike some biology-based anticancer strategies [1, 15, 16, 19], use of a DDV is implementable with nononcolytic viruses and, therefore, avoids the dangers [19] of using onco-

In practice, we have discovered a phage T3 capsid that appears to have DDVfavorable characteristics needed for implementation of our strategy. First, phage T3 (and presumably its capsid) has recently been found to have exceptionally high persistence in mouse blood (3–4 h), unlike the T3 relative, T7 [30]. Second, one empty, but otherwise phage-like T3 capsid, is impermeable (for over 20 years) to the compound, Nycodenz (821 Da molecular weight) (reviewed in [31]). But, when the temperature is raised to 45°C, Nycodenz enters this capsid [32], presumably through a gate that opened. The concept is that, if we adapt this capsid to use as gated DDV, some of the needed engineering has already been done by natural

Phages T3 and T7 are illustrated at the left in **Figure 1**. The gated capsid is illustrated at the right in **Figure 1**. This capsid is generated during DNA packaging that had been initiated by a DNA-free procapsid called capsid I (not shown). During packaging, capsid I expands and becomes the more angular and stable capsid (capsid II) illustrated in **Figure 1**. In nature, the capsid-gate is a ring of 12 gp8 molecules (**Figure 1**) that acts as entry portal for DNA during DNA packaging [31–33]. Most T3 and T7 capsid II particles are purified after having detached from DNA during infected cell lysis. The amount is 5–10 mg capsid II per liter of culture. The last purification step is buoyant density centrifugation in a Nycodenz or Metrizamide density gradient. Nycodenz low density (NLD) capsid II has the gp8 "gate" and is impermeable to Nycodenz and Metrizamide. The low density (1.08 g/ml) is caused

by high internal hydration, which is caused by Nycodenz impermeability.

The EPR effect is the spontaneous accumulation of nanoparticles in tumors, observed for an uncharacterized phage in 1940 [18] (reviewed in [19]). The causes of the EPR effect are (1) porosity of tumor blood vessels and relative tightness of healthy blood vessels, so that nanoparticles enter tumors, but usually not healthy tissue and (2) poor tumor lymphatic drainage, so that nanoparticles remain [20–25]. The EPR effect is the basis for the use of several FDA-approved, drug-

**14**

*Phages T3 and T7 (left); and T3 and T7 NLD capsid II (right). The graphic legend at the top indicates the various capsid components. A protein is labeled by gp, followed by the number of the encoding gene. Analogous T3 and T7 genes have the same numbers. If NLD capsid II is used as DDV, the perimeter of the DDV is defined by the gp10 shell; the gate is the gp8 portal. All structures are the same for phage T3 as they are for phage T7 (reviewed in [31, 32]).*

Nycodenz high density (NHD) capsid II is Nycodenz-permeable (1.28 g/ml) and is completely separated from NLD capsid II during buoyant density centrifugation in a Nycodenz or Metrizamide density gradient [31, 33–35]. We use NHD capsid II as a control during loading experiments.

In the case of T3 NLD capsid II, gated entry of Nycodenz has been observed via raising of temperature. The likely entry channel was the axial hole of the gp8 ring. This conclusion was drawn, in the case of T7 NLD capsid II, from (1) entry kinetics of the fluorescent dye, bis-ANS [1,1′-bi(4-anilino)naphthalene-5,5′-di-sulfonic acid; 673 Da], and (2) covalent cross-linking of bis-ANS to channel proteins [33]. Asymmetric reconstruction-cryo-EM [36] revealed that obstruction of the T7 gp8 channel (and presumably the T3 channel) varies.

In support of working to implement the above gating-based strategy, the following quote from 2005 presents an expert opinion of what is needed for the next generation of anticancer DDVs [27]. "An ideal liposomal anticancer drug would exhibit little or no drug release while in the plasma compartment, thus ensuring limited exposure of the drug to healthy tissue. This feature would also maximize drug delivery to disease sites, as mediated by the movement of the drug-loaded liposomes from the plasma compartment to the extravascular space at disease sites, such as a region of tumor growth. Following localization, however, the drug-loaded liposome must transform itself from a stable carrier to an unstable carrier. This would ensure that the drug, which has localized in the diseased site, is bioavailable." To our knowledge, no details for such "controlled release" via a liposomal DDV have been published. The system described here is designed to accomplish what is described in the above quote. However, implementation uses gating of a DDV, not programmed instability of a DDV.

To proceed further, we need a procedure for rapidly determining whether a drug is loaded in a capsid. In the current study, we have developed native agarose gel electrophoresis (AGE) for this purpose. The detection is performed via capsid band fluorescence produced by the compound loaded.
