2. Medical applications of VLPs

#### 2.1 Gene therapy

The VLPs have been largely studied for their use in gene therapy because of their natural capacity to transport nucleic acids and to integrate these genes into the host genome. Mainly, the nanoparticles come from virus of mammals since they have the intrinsic capacity to internalize into human or animal cells [8].

The family of adenovirus is the most used virus to produce VLPs for gene therapy. They are icosahedral capsids containing double chain DNA. So far, 24% of the clinical tests for gene therapy are using this kind of virus. These viral vectors reach a load capacity up to 35 kilobases of nonviral DNA that allow the incorporation of big transgenes as well as regulation elements [9].

The VLPs show several advantages; they are easily produced with high viral titers, they have the transduction capacity with high efficiency in both growing and quiescent cells, great genome stability, low levels of viral genome integration, and good biological characterization [10].

The surface of VLPs can be chemically modified with polymers such as polyethylene glycol (PEG) and poly-N-(2-hydroxypropyl) methacrylamide (poly-HPMA) with the aim to decrease their immunogenicity, avoiding a fast elimination. On the other hand, the chemical modification of the VLP's surface could be used to bind ligands for specific cell receptors to be internalized in targeted tissues [11].

#### 2.2 Drug delivery

In addition to gene transport to targeted cells for gene therapy, the VLPs are able to deliver to specific tissues small molecules as therapeutic agents. The goal is to deliver the therapeutic drug to the tissues, which is needed to increase the treatment efficiency and importantly to reduce the doses and thus the side effects. The

encapsulated drug is not systemically present in the body, is protected against degradation, and increases its biocompatibility [12].

The enzymes could be also encapsulated inside the VLPs and used as therapeutic agents; however, their potential has not been fully studied because some limitations include biodegradation susceptibility by proteases and nonspecificity to be targeted and internalized by specific cells. These disadvantages could be solved by encapsu-

The enzyme cytosine deaminase from yeast was encapsulated inside the VLPs from the monkey virus 40 (SV40) belonging to the polyomavirus family. This enzyme is able to transform the prodrug 5-fluorocytosine to the active drug 5 fluorouracil that induces cell death. The monkey CV-1 cells were treated with the

fluorocytosine due to its transformation to 5-fluorouracil. This is just one example of the potential applications of VLPs as carriers for enzymatic activity targeted to a

Diver compounds are widely used for medical (in vivo) imaging for diagnostic and therapeutic treatment evaluation. The VLPs have been studied in this field because they could be functionalized, inside and outside, with multiple contrast molecules such as fluorophores (including fluorescent proteins), quantum dots, as well as certain metals. In addition, functionalized VLPs can be directed and accumulated in specific targeted tissues. Both characteristics enhance the specificity and biocompatibility of medical imaging techniques [20]. The immobilization of fluorophores on VLPs allows a high loading in a site-specific fashion to prevent aggregation and lowering the quenching of fluorescent compounds [21].

An interesting example of the use of VLPs for in vivo imaging was reported by Hooker et al. [22]. The VLPs from the phage MS2 were chemically conjugated with Gd3+ ions, a contrast agent used in nuclear magnetic resonance (NMR), which is a noninvasive technique widely used for the diagnostic of several diseases. In this work, the functionalization of the virus-like particles was carried out in both internal and external phases of the protein nanoparticle. The modified nanoparticles showed an improved solubility and stability, and an important increase in the fluorescence relaxation that allowed an increase in the sensibility of the technique. Functionalized VLPs with fluorescent proteins have been synthesized to elucidate the infection and pathogenesis of some viruses. The capsid protein VP2 from the human parvovirus B19 conjugated in the external surface with the enhanced green fluorescent protein (EGFP) was able to be internalized in tumor cells and, through the microtubule network, reach the nucleus (Figure 4) [23]. These nanoparticles, in addition, could be useful to understand the biology of the virus infections as they could be monitored to elucidate the virus in vivo behavior.

Another application in which the VLPs are gaining interest is as biosensors for the detection of DNA, toxins from pathogens, or protein disease markers. The virus-like nanoparticles act as carriers of different ligands, receptors, or reporter molecules to obtain sensors with high specificity and sensitivity for the in vitro diagnosis [24]. The VLPs have been used mainly in two different techniques,

With the aim to increase the microarray sensitivity in samples with low DNA concentration, the VLPs from the Cowpea mosaic virus (CPMV) were functionalized

NeutrAvidin (NA), a compound able to recognize biotin. The conjugation was made

with 42 molecules of carbocyanine fluorophore (Cy5) and one molecule of

VLPs containing cytosine deaminase, and the cells were sensible to the 5-

lating the enzymes inside the VLPs as discussed later.

specific tissue [19].

Viral Structures in Nanomedicine

DOI: http://dx.doi.org/10.5772/intechopen.90099

2.3 Medical imaging

2.4 Diagnosis

5

microarrays, and immunoassays.

There is abundant information on the drug encapsulation into VLPs and also drug binding on the capsid surface, especially for chemotherapy [13–15]. In an interesting work by Bar et al. [16], a filamentous phage was chemically modified with the chemotherapeutic compound hygromycin or genetically modified with doxorubicin to treat cancer. The strategy for the phage modification with doxorubicin included a mutation by genetic engineering the N-end of the main coat protein (p8) introducing a peptide sensible to be degraded by the cathepsin B that was conjugated with the doxorubicin. With the goal to drive the VLPs to the targeted tissues, the VLPs were functionalized with three different IgG antibodies that recognize specific cell receptors on tumor cells. This functionalized nanocarrier containing the chemotherapeutic agent was effectively recognized and internalized in the tumor cells, liberating the drug by the hydrolyzation of the peptide by the cathepsin B from liposomes and inducing the cellular death. The multivalent VLP was able to deliver 3500 molecules of the drug per capsid, inducing a significant enhancement of the inhibitory efficiency on the tumor cells.

The photosensitizers are molecules with biomedical interest for targeted photodynamic therapy (TPD). These molecules are excited by light at specific wavelengths and produce reactive oxygen species (ROS) that are able to kill tumor cells [17].

Nanoparticles from the Cowpea chlorotic mottle virus (CCMV) were doubly functionalized to kill bacteria by TPD [18]. The VLPs were covalently modified with ruthenium complex as photosensitizer (Figure 3) and directed through antibodies to pathogen bacteria Staphylococcus aureus, which is able to produce biofilms. The functionalized VLPs were then irradiated with light at 470 nm producing reactive oxygen species.

#### Figure 3.

The cysteines introduced by site-directed mutagenesis on the surface of the VLP from CCMV allow the chemical conjugation of the photosensitizer (modified from [18]).

#### Viral Structures in Nanomedicine DOI: http://dx.doi.org/10.5772/intechopen.90099

The enzymes could be also encapsulated inside the VLPs and used as therapeutic agents; however, their potential has not been fully studied because some limitations include biodegradation susceptibility by proteases and nonspecificity to be targeted and internalized by specific cells. These disadvantages could be solved by encapsulating the enzymes inside the VLPs as discussed later.

The enzyme cytosine deaminase from yeast was encapsulated inside the VLPs from the monkey virus 40 (SV40) belonging to the polyomavirus family. This enzyme is able to transform the prodrug 5-fluorocytosine to the active drug 5 fluorouracil that induces cell death. The monkey CV-1 cells were treated with the VLPs containing cytosine deaminase, and the cells were sensible to the 5 fluorocytosine due to its transformation to 5-fluorouracil. This is just one example of the potential applications of VLPs as carriers for enzymatic activity targeted to a specific tissue [19].

#### 2.3 Medical imaging

encapsulated drug is not systemically present in the body, is protected against

There is abundant information on the drug encapsulation into VLPs and also drug binding on the capsid surface, especially for chemotherapy [13–15]. In an interesting work by Bar et al. [16], a filamentous phage was chemically modified with the chemotherapeutic compound hygromycin or genetically modified with doxorubicin to treat cancer. The strategy for the phage modification with doxorubicin included a mutation by genetic engineering the N-end of the main coat protein (p8) introducing a peptide sensible to be degraded by the cathepsin B that was conjugated with the doxorubicin. With the goal to drive the VLPs to the targeted tissues, the VLPs were functionalized with three different IgG antibodies that recognize specific cell receptors on tumor cells. This functionalized nanocarrier containing the chemotherapeutic agent was effectively recognized and internalized in the tumor cells, liberating the drug by the hydrolyzation of the peptide by the cathepsin B from liposomes and inducing the cellular death. The multivalent VLP was able to deliver 3500 molecules of the drug per capsid, inducing a significant

degradation, and increases its biocompatibility [12].

Technology, Science and Culture - A Global Vision, Volume II

enhancement of the inhibitory efficiency on the tumor cells.

cells [17].

Figure 3.

4

conjugation of the photosensitizer (modified from [18]).

reactive oxygen species.

The photosensitizers are molecules with biomedical interest for targeted photodynamic therapy (TPD). These molecules are excited by light at specific wavelengths and produce reactive oxygen species (ROS) that are able to kill tumor

Nanoparticles from the Cowpea chlorotic mottle virus (CCMV) were doubly functionalized to kill bacteria by TPD [18]. The VLPs were covalently modified with ruthenium complex as photosensitizer (Figure 3) and directed through antibodies to pathogen bacteria Staphylococcus aureus, which is able to produce biofilms. The functionalized VLPs were then irradiated with light at 470 nm producing

The cysteines introduced by site-directed mutagenesis on the surface of the VLP from CCMV allow the chemical

Diver compounds are widely used for medical (in vivo) imaging for diagnostic and therapeutic treatment evaluation. The VLPs have been studied in this field because they could be functionalized, inside and outside, with multiple contrast molecules such as fluorophores (including fluorescent proteins), quantum dots, as well as certain metals. In addition, functionalized VLPs can be directed and accumulated in specific targeted tissues. Both characteristics enhance the specificity and biocompatibility of medical imaging techniques [20]. The immobilization of fluorophores on VLPs allows a high loading in a site-specific fashion to prevent aggregation and lowering the quenching of fluorescent compounds [21].

An interesting example of the use of VLPs for in vivo imaging was reported by Hooker et al. [22]. The VLPs from the phage MS2 were chemically conjugated with Gd3+ ions, a contrast agent used in nuclear magnetic resonance (NMR), which is a noninvasive technique widely used for the diagnostic of several diseases. In this work, the functionalization of the virus-like particles was carried out in both internal and external phases of the protein nanoparticle. The modified nanoparticles showed an improved solubility and stability, and an important increase in the fluorescence relaxation that allowed an increase in the sensibility of the technique.

Functionalized VLPs with fluorescent proteins have been synthesized to elucidate the infection and pathogenesis of some viruses. The capsid protein VP2 from the human parvovirus B19 conjugated in the external surface with the enhanced green fluorescent protein (EGFP) was able to be internalized in tumor cells and, through the microtubule network, reach the nucleus (Figure 4) [23]. These nanoparticles, in addition, could be useful to understand the biology of the virus infections as they could be monitored to elucidate the virus in vivo behavior.

#### 2.4 Diagnosis

Another application in which the VLPs are gaining interest is as biosensors for the detection of DNA, toxins from pathogens, or protein disease markers. The virus-like nanoparticles act as carriers of different ligands, receptors, or reporter molecules to obtain sensors with high specificity and sensitivity for the in vitro diagnosis [24]. The VLPs have been used mainly in two different techniques, microarrays, and immunoassays.

With the aim to increase the microarray sensitivity in samples with low DNA concentration, the VLPs from the Cowpea mosaic virus (CPMV) were functionalized with 42 molecules of carbocyanine fluorophore (Cy5) and one molecule of NeutrAvidin (NA), a compound able to recognize biotin. The conjugation was made

To increase the efficiency of immunoassays for the diagnosis of some diseases, highly sensible systems are necessary. To obtain this, the capsids of the hepatitis B virus have been genetically modified to express a fragment of the protein A from Staphylococcus (SPAB) in the coat protein surface. This fragment has specific affinity

nanoparticles, the right orientation of antibodies with the specific variable antigen domains was fully accessible to recognize the marker proteins such as troponin, which is a protein found in the patients showing damage in the cardiac muscle that show high tendency to have heart attacks. The use of these VLPs together with the functionalized plates with PVDF membranes or nickel nanostructures allowed the sensitivity increase of the technique up to attomolar level (10<sup>18</sup> M). This is six

The treatment of disorders originating from enzymatic activity deficiencies using therapeutic enzymes was first described 50 years ago [27]. There are many diseases that originated from the lack of one or more enzymatic activities, and because of this, in some cases, the administration of exogenous enzymes has been successfully used as enzyme replacement therapy (ERT). Enzyme replacement therapy has been recently reviewed [28–30]. The success of ERT is mainly based on enzyme biochemistry, local enzyme concentrations, the ability of treated cells to turn over and be replaced by normal cells, and the substrate movement, redistribution, and storage. All these factors could be modulated and improved by the use of

Comellas-Aragonès et al. [31] developed a virus-based single-enzyme nanoreactor. Horseradish peroxidase was encapsulated in the Cowpea chlorotic mottle virus, and the enzymatic behavior of the developed nanoreactor was studied. It was the first report that showed the permeability of virus capsid for substrate/ product and the alteration of permeability by the change of pH. A breakthrough in VLP-based nanoreactors was achieved by Patterson et al. [32] who constructed a densely packed multienzyme system able to perform a coupled cascade of reactions. In this work, a 160-kDa protein was produced by the fusion of β-glucosidase, galactokinase, and glucokinase and encapsulated into bacteriophage P22-derived capsids. In addition, their results showed that intermediate channeling between sequential enzymes is dependent on both inter-enzyme distance and a balance

Sánchez-Sánchez et al. [33] effectively encapsulated a cytochrome P450 (CYP) variant from Bacillus megaterium to VLPs constituting of coat protein from Cowpea chlorotic mottle virus (Figure 6). These catalytic VLPs were able to transform the chemotherapeutic prodrug tamoxifen into active products similar to those obtained with human CYP. In a subsequent study, Sánchez-Sánchez et al. [34] encapsulated CYP into bacteriophage P22-derived capsids. Nanobioreactors contained 109 molecules of CYP per capsid and enzyme stability toward protease degradation, and acidic pH was increased. Furthermore, these nanobioreactors were internalized into human cervix carcinoma cells, and as expected, P22-CYP transfected cells showed a 10-fold higher CYP activity than nontreated cells. PEGylation of the VLP capsids strongly reduced or eliminated the immunogenic response, and by functionalizing

specific ligands to the free end of the PEG molecules, the virus-derived nanoreactors could be targeted to receptors on tumor cells [35, 36]. Ligandreceptor-mediated cell internalization increased CYP activity inside cells and thus

to the Fc domain of the immunoglobulins (IgG). With these virus-like

orders of magnitude lower than the conventional procedure [26].

3. Biocatalytic nanoreactors for enzyme therapies

between the kinetic parameters of the enzymes involved.

virus capsids as carriers.

Viral Structures in Nanomedicine

DOI: http://dx.doi.org/10.5772/intechopen.90099

7

#### Figure 4.

Confocal microscopy of Hep G2 cells incubated with virus-like nanoparticles VP2-EGFP. (A) Immuno-dyeing with VP2 antibodies visualized with Alexa-633 antimouse after 6-h incubation. The EGFP was directly visualized. (B) Immuno-dyeing with anti-α-tubulin visualized with Alexa-633 antimouse after 4-h incubation. The colocalization of VLP VP2-EGFP and microtubules is shown in yellow (modified from [23]).

#### Figure 5.

Comparative scheme of the detection method with NA-Cy5-CPMV and the conventional method with streptavidin-Cy5 (modified from [25]).

through cysteines introduced by genetic engineering. The nanoparticle NA-Cy5- CPMV was then used for the detection of genes from the pathogen bacteria Vibrio cholerae O139 by a microarray assay. These nanoparticles bind specifically the biotined DNA through the NA recognition (Figure 5). Due to the high emitted fluorescence by the Cy5 molecules, is possible to detect very low concentrations of DNA as 1 to 10 copies of the genome [25].

#### Viral Structures in Nanomedicine DOI: http://dx.doi.org/10.5772/intechopen.90099

To increase the efficiency of immunoassays for the diagnosis of some diseases, highly sensible systems are necessary. To obtain this, the capsids of the hepatitis B virus have been genetically modified to express a fragment of the protein A from Staphylococcus (SPAB) in the coat protein surface. This fragment has specific affinity to the Fc domain of the immunoglobulins (IgG). With these virus-like nanoparticles, the right orientation of antibodies with the specific variable antigen domains was fully accessible to recognize the marker proteins such as troponin, which is a protein found in the patients showing damage in the cardiac muscle that show high tendency to have heart attacks. The use of these VLPs together with the functionalized plates with PVDF membranes or nickel nanostructures allowed the sensitivity increase of the technique up to attomolar level (10<sup>18</sup> M). This is six orders of magnitude lower than the conventional procedure [26].
