3. Biocatalytic nanoreactors for enzyme therapies

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 virus capsids as carriers.

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 between the kinetic parameters of the enzymes involved.

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

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

Comparative scheme of the detection method with NA-Cy5-CPMV and the conventional method with

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]).

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

DNA as 1 to 10 copies of the genome [25].

streptavidin-Cy5 (modified from [25]).

Figure 4.

Figure 5.

6

photodynamic therapy (PDT) by Chauhan et al. [36]. The P22 bacteriophage encapsulating CYP activity was multifunctionalized with a porphyrin-based photosensitizer for PDT effect and estradiol-based ligand for targeted delivery to ER+ breast tumor cells (Figure 7). The system was able to generate reactive oxygen species upon illumination with light that in synergy with CYP activity transformed tamoxifen (prodrug) to 4-hydroxytamoxifen (active drug) and showed approximately threefold higher toxicity than nontargeted VLPs. Additionally, the PEG-coat of the multifunctional nanoreactors rendered them invisible to macrophages and treatment of tumor cells with these nanoreactors significantly reducing prodrug dosage. Results obtained indicate that a drastic reduction of chemotherapy side effects and an increase in treatment effectiveness could be expected. The study is the first example to show the applicability of the VLP platform as a combinatory treatment modality, inhibiting tumor cells in multiple ways at once, which will be advantageous to overcome tumor heteroge-

Finally, Schoonen et al. [37] encapsulated T4 lysozyme (T4L) inside elastin-like polypeptide (EPL)-stabilized CCMV capsids. The activity of T4L is highly dependent on the salt concentration and pH of the environment under which these capsids are not able to form; however, by adding metal ions, the system was stabilized. Four T4L molecules per CCMV capsid were encapsulated and remained catalytically active. Their work opened the possibility of utilizing shielded T4L in

Bionanotechnology, as an emerging technological field, has opened a vast number of potential applications, in which biomedical applications are included. VLPs carrying different cargos could be effectively functionalized with different ligands to be recognized and cell-internalized for tissues containing the specific receptor. In spite of several advantages showed by the VLPs in the biomedical field, still there are some challenges to be solved to be practically used. These challenges are mainly related to the immunogenic response. Different strategies could be implemented to avoid the immune system recognition that includes the covering of the VLPs with a polymer such as polyethylene glycol or the genetic engineering of the viral coat proteins to mutate the epitopes more immunogenic [13]. The recent advancements in the bioinspired "active" stealth covers combining the property to reduce or eliminate the immunogenic response, and the active

targeting, could be beneficial for treating diseased tissue via a biomimicry approach with lower side effects. The toxicity of VLPs should be also evaluated with emphasis on the fate of these nanoparticles in the organism and their possible

Another problem to be solved is the production of VLPs at large scale. Some VLPs can be easily produced at large scale, while others are hard to be expressed and purified. Research efforts are still necessary to have efficient systems of

We can conclude so far that the use of VLPs for biomedical and therapeutic purposes is still in its infancy but shows enormous potential, and thus research efforts are still needed. However, it is clear that the VLPs are excellent systems for several applications in the biomedical field including drug, enzyme and gene delivery, medical imaging, and biosensors. These virus-derived nanoparticles are

4. Challenges for the use of VLPs in nanomedicine

heterologous expression of coat proteins of VLPs [39].

promising candidates for the treatment and diagnosis of diseases.

neity and reoccurrence issues.

Viral Structures in Nanomedicine

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

antibiotic applications.

side effects [38].

9

#### Figure 6.

Bionanoreactor of cytochrome P450 encapsulated in virus-like particle for the activation of prodrug tamoxifen to the active drug for breast cancer treatment.

#### Figure 7.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

increased tamoxifen cell sensitivity of both human cervix and breast tumor cells, reducing the dose needed to kill these cells by 50% [35].

The multivalency and versatility of virus-derived nanovehicles were also used for targeted enzyme delivery in conjunction with the synergistic effect of

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

photodynamic therapy (PDT) by Chauhan et al. [36]. The P22 bacteriophage encapsulating CYP activity was multifunctionalized with a porphyrin-based photosensitizer for PDT effect and estradiol-based ligand for targeted delivery to ER+ breast tumor cells (Figure 7). The system was able to generate reactive oxygen species upon illumination with light that in synergy with CYP activity transformed tamoxifen (prodrug) to 4-hydroxytamoxifen (active drug) and showed approximately threefold higher toxicity than nontargeted VLPs. Additionally, the PEG-coat of the multifunctional nanoreactors rendered them invisible to macrophages and treatment of tumor cells with these nanoreactors significantly reducing prodrug dosage. Results obtained indicate that a drastic reduction of chemotherapy side effects and an increase in treatment effectiveness could be expected. The study is the first example to show the applicability of the VLP platform as a combinatory treatment modality, inhibiting tumor cells in multiple ways at once, which will be advantageous to overcome tumor heterogeneity and reoccurrence issues.

Finally, Schoonen et al. [37] encapsulated T4 lysozyme (T4L) inside elastin-like polypeptide (EPL)-stabilized CCMV capsids. The activity of T4L is highly dependent on the salt concentration and pH of the environment under which these capsids are not able to form; however, by adding metal ions, the system was stabilized. Four T4L molecules per CCMV capsid were encapsulated and remained catalytically active. Their work opened the possibility of utilizing shielded T4L in antibiotic applications.
