A Plant Virus Nanoparticle Toolbox to Combat Cancer

Nowadays, nanoparticles are a popular toolbox used in cancer research for improving the pharmaceutical capacity of therapeutic agents. Viral nanoparticles (VNPs) are naturally-occurring NPs that have emerged as key players. VNPs are structures between 30 -200 nm in diameter, having unique size- and shape-dependent physicochemical and biological properties. Depending on their origin, VNPs can belong to mammalian viruses, bacteriophages, or plant viruses. While VNPs exist with differences in shape, structure, and molecular components, they can share similar applications in cancer therapy. VNPs offer powerful platforms for vaccines, immunotherapy, and delivery of theranostic payloads. Due to cytotoxicity, unwanted immunologic reactions and adverse side effects, mammalian viruses influence VNP technology’s cost, complexity, and safety. With regard to these limitations, plant viruses or plant viral nanoparticles (PVNPs) have emerged as an alternative platform. The many properties of PVNPs make them good candidates for developing tumour therapy. These features include their lack of pathogenicity, biocompatibility and biodegradability properties in mammalian systems, stability in rigid environment conditions, simple external functionalisation by either single or multiple functional group expression, payload loading capacity and their inherent immunostimulatory effect. PVNPs include Brome mosaic virus (BMV), Red clover necrotic mosaic virus (RCNMV), Cowpea chlorotic mottle virus (CCMV), Cowpea mosaic virus (CPMV) Potato virus X (PVX), Tobacco mosaic virus (TMV), Alfalfa mosaic virus (AMV) that have been used as scaffolds in cancer research. Here, we present the functions and applications of PVNPs in cancer treatment.

Plant virus nanoparticle-based tumour therapies

PVNPs are potent platforms from the nano-toolbox for the treatment of cancer. Similar to synthetic NPs, the nature, structure, and physicochemical features of PVNPs determine their medical application. PVNPs are self-assembled protein coat (CP) units that form a hollow structure for entrapping nucleic acids. In the presence of nucleic acid, CPs self-assemble in icosahedral, filamentous, rod-like, and bacillus morphologies. In contrast, in the deprivation of nucleic acids, CPs self-assemble as hollow virus-like particles (VLP), or spherical nanoparticles (SNP). PVNPs used to combat cancer can manifest as two strategies: 1) as protein nanovehicles for loading anticancer therapeutic agents to increase their therapeutic efficacy, and 2) as immunomodulatory agents for enhancing anti-tumour immune responses.

The unique structural and chemical properties of PVNPs make them ideal nanocarriers. PVNPs’ empty internal cavities, surface groups, and open/closed conformations enable cargo to be loaded. Therapeutic cargos are usually loaded in PVNPs via noncovalent (i.e., self-assembly, infusion, and charge/ ionic interaction) and covalent (i.e., genetically and chemically) mechanisms (reviewed by refs). Genetic manipulation is often used for displaying target ligands, antigenic structures, or specific amino acids or small peptides as tags (e.g. SNAP-tag) for further functionalisation. The self-assembly process is based on the caging of coat protein around a cargo. PVNP chemical modifications can be achieved through conjugation reactions (bioconjugate chemistry, click chemistry). Some PVNPs can trap cargo via a pore-opening mechanism in response to environmental conditions (i.e., pH and salt concentrations). PVNPs possess a negative/positive charge within a biological pH, and thus, they load charged cargos via electrostatic interactions.

More importantly and for addressing human solid tumours, PVNPs can target and transfer their cargo into the tumour microenvironment (TME) and within tumour cells themselves. Accumulation of PVNPs in TME is determined by size and blood distribution. PVNPs tend to accumulate in TME much more than in normal tissue, because of leaky vasculature and poor lymphatic drainage, as well as enhanced permeability and retention (EPR) effect. For targeting tumour cells, PVNPs don't require specific ligands by nature, however, they could be manipulated or engineered to display agonist ligands of tumour cell-membrane receptors, in order to directly deliver and transfer their cargo. Generally, structural properties (e.g., size, charge, and shape), various methods of cargo loading, and bioengineering all provide PVNPs for non-targeted and targeted delivery of therapeutic agents (e.g., small molecule drugs, nucleic acids, peptides, and proteins) and immunotherapeutic agents for cancer treatment (Figure 1).

PVNPs for the delivery of therapeutic agents

Therapeutic agents consisting of chemical and biological drugs can target and kill tumour cells via specific mechanisms. Traditional therapeutic drugs are highly effective to kill cancerous cells. However, their systemic administration have certain disadvantages such as lower bioavailability, minimal effectiveness, and severe side effects. Therefore, PVNP-based formulations have been designed as nano vehicles toward improving the pharmacological profiles of therapeutic drugs (Table 1). As mentioned above, PVNPs can load therapeutic agents via the exterior and/or interior of the capsid surface using covalent or non-covalent interactions. The nanoparticulate features (size, shape, charge, and surface functionalities) of PVNPs, and leaky nature of the vasculature (or EPR effect) of tumours lead to the accumulation of loaded PVNPs with therapeutic agents in the TME.

For example, charge-driven drug loading strategies were applied for encapsulating mitoxantrone (MTO) into TMV, a 300 × 18 nm nanorod containing a 4 nm-wide channel, and lined with glutamic acids. The negative charge of the glutamic acids allows for electrostatic interactions with the positively charged MTO, thus allowing for pH dependent drug-loading and release. In vitro and in vivo results confirmed that MTO maintained its efficacy when delivered by TMV in a panel of cancer cell lines in addition to a triple negative breast cancer mouse model. Another drug for loading into the nano-channel of TMV has been the active dictation form of cisplatin (cisPt2+), making use of the negatively charged Glu acid side chains that line the interior channel of TMV. TMV-cisPt exhibited superior efficacy vs free cisPt in ovarian tumour mouse models.

The greatest challenge in PVNP -based therapeutic agents is the low efficiency of delivery to tumour cells. PVNP-based targeted delivery is designed using an overexpression of tumour cell biomarkers -based agonist ligands for targeting, binding and delivering the payload to tumour cells. For example, display of (((S)-5-amino-1-carboxypentyl) carbamoyl)-L-glutamic acid (DUPA), a specific ligand to prostate-specific membrane antigen (PSMA), in TMV and loaded with MTO increased cytotoxicity of PSMA+ prostate cancer threefold, and is a promising therapeutic strategy. PVNPs displaying folic acid, GE11 (a small peptide with 12 amino acids), HER2 ligands (trastuzumab, CH401 epitope), tumour-homing peptides (THPs, IR780 iodide, F3), TRAIL (tumour-necrosis factor related apoptosis-inducing ligand), arginine–glycine–aspartate (RGD) peptide, and Asp-Gly-Glu-Ala (DGEA) peptide, have been shown to selectively target tumour cells.

PVNPs for the delivery of phototherapy agents

Photodynamic therapy (PDT) and photothermal therapy (PTT) are promising avenues for improving the efficacy of cancer treatment. PVNPs can deliver photo activated therapeutic agents for inducing cytotoxicity via PDT and PTT. In this process, PD-PVNP accumulates in TME, and then is activated by light to generate reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radicals, superoxide anions or singlet oxygen, which consequently cause cytotoxicity to tumour cells. For example, the cationic photosensitiser Porphyrin encapsulated the interior channel of TMV via electrostatic interactions to improve cell uptake and efficacy compared to free photosensitisers in a melanoma model. In another study, the incorporation of Zn-EpPor PS through electrostatic interactions with the carboxyl dendron attached to CPMV improved 2-fold the uptake efficacy and cell death in a B16F10 melanoma cell line compared to free PS. Free Zn-Por3+ displayed the greatest cell toxicity and the loading of Zn-Por into TMV and TMGMV resulted in slightly decreased cell toxicity in vitro. In contrast, targeted Zn-Por -TMV (with the nucleolin-specific F3 peptide accumulating at the cancer cell surface) can increase tumour cytotoxicity fivefold.

Photothermal therapy (PT) includes a PVNP associated with a photothermal agent. In this process, PT-PVNP accumulates in TME, and becomes activated by light to generate heat. Gd-TMV– polydopamine (PDA), with a strong near-infrared absorption and a high photothermal conversion efficiency (28.9%), offer promising results for effectively killing PC-3 prostate cancer cells in vivo and in cancer models. Coating of TMV with polydopamine (PDA), as a PTT agent was demonstrated to increase anti-tumour efficacy based on PTT- immunotherapy in B16F10 dermal melanoma in C57BL/6 mice. Targeted Cowpea chlorotic mottle virus (CCMV) capsids with tumour homing peptide F3 (via genetic engineering), and loaded with near-infrared fluorescent dye IR780 iodide, (F3-CCMV-IR780 NPs), displayed excellent molecular targeting PTT to nucleolin receptor over-expressed on the surface of MCF-7 tumour cells.

PVNPs for the delivery of immunotherapeutic agents

Nanocarrier properties of PVNPs have prepared them for the delivery of immunotherapeutic agents to the target site (e.g. TME, antigen presenting cells (APCs), and other components of the immune system) to improve the therapeutic index. Toward this goal, encapsulation of oligodeoxynucleotides (CpG ODNs, ODN1826), as agonists of Toll-like receptor 9 (TLR 9) into CCMV and TLR 7 agonist (1V209) into TMV has been shown to slow tumour growth and prolong survival in mouse models of colon cancer and melanoma. This demonstrated that bioconjugation with the anti-PD-1 peptide SNTSESF (AUNP) into CPMV (the CPMV-AUNP formulation) increased the anti-tumour efficacy of AUNP into ovarian cancer cells compared to free peptide.

PVNPs structural properties (i.e., size, shape and rigidity) can transfer and stimulate APCs within the lymph node. For example, to overcome immunological tolerance against HER2-positive tumour cells, integrated HER2 epitopes loaded onto Potato virus X (PVX), and CPMV acted as vaccines without requiring additional adjuvants to induce a strong and sustained anti-HER2 immune response. Similarly, a VLP-based vaccine has been designed via click chemistry with the attachment of the HER2-derived CH401 peptide epitope into PhMV. Results have shown that PhMV-based vaccine enhanced anticancer immunity by high titers of HER2-specific immunoglobulins, increased the toxicity of antisera to DDHER2 tumour cells, and prolonged survival of the vaccinated vs. naïve BALB/C mice. Testis antigen NY-ESO-1 is an attractive antigenic target for cancer vaccines. Displaying multiple copies of human HLA-A2 restricted peptide antigen NY-ESO-1157–165 into CPMV enhances uptake and activation of APCs and stimulates a potent CD8+ T cell response. This study shows the potential of CPMV-NY-ESO-1 vaccine against NY-ESO-1+ malignancies. These studies also explain how PVNP formulations are expected to exhibit prolonged tumour residence and favorable intratumoural distribution. (Table 1)

PVNPs for in situ vaccination (ISV)

The inherent immunogenicity of PVNPs have provided them with tremendous potential as direct immunomodulators to activate the innate immune response. The function of PVNPs in immunomodulation depends on their structural components, capsid protein and genome. They can act as non-self (foreign), or danger signals, and active pattern recognition receptors (PRRs) on immune cells carrying Toll like receptors (TLRs). For example CPMV (virion) and empty CPMV (eCPMV, without the nucleic acid) capsids are recognised by MyD88-dependent TLR2 and TLR4, and the release of the ssRNA contained within CPMV and PapMV is recognized by TLR7. It was demonstrated that PVNPs can act as pathogen-associated molecular patterns (PAMPs) for TLR of the surface (1, 2, 4, 5, and 6) or the endosome (TLRs 3, 7, 8, and 9) on APCs.

In situ vaccination (ISV) uses intratumoural injection of PVNPs to activate the innate and adaptive immune system in TME. Virions with active or deactivated nucleic acids and VLP can be used for ISV. PVNPs-ISV therapy leads to changes in cytokine levels within the TME, and reprogram and repolarise suppressed innate immune cells toward an anti-tumour phenotype. They also induce the recruitment of innate immune cells that are cytotoxic to cancer cells. PVNPs can induce pro-inflammatory cytokine production such as interleukin (IL)-1, IL-6 and IL-12, interferon (IFN)-, and IFN- that potentiate to induce an adaptive immune response (Figure 2). A typical PVNP used for ISV is CPMV. The CPMV capsid triggers TLRs 2, 4 and the ssRNA within CPMV activates TLR 7, and receptor signalling cascades lead to the release of immunostimulatory cytokines such as IL-1, IL-12, IFN-, chemokine ligand 3, macrophage inflammatory protein-2, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

The tumour acts as a resource of antigens in ISV, and upon tumour antigen release in the TME, processing and priming by the APCs leads to the activation of the induction of systemic and tumour-specific immune responses. To date, the in situ injection of CPMV, Cowpea severe mosaic virus (CPSMV) and Tobacco ring spot virus (TRSV), Cowpea Chlorotic mottle virus (CCMV), Physalis mosaic virus (PhMV), and Sesbania mosaic virus (SeMV), TMV, PVX, Papaya mosaic virus (PapMV), Alfalfa mosaic virus (AMV) have demonstrated anti-tumour potentials in mouse models.

PVNP-mediated ISV is generally effective only against small tumours, and most patients do not respond to PVNP monotherapy. Combining multiple treatment regimens with PVNP -based ISV could form the basis for success. For example, immune checkpoint therapy (ICT) has the potential to treat cancer by removing the immunosuppressive brakes on T cell activity. It is shown that combined treatment with CPMV and selected checkpoint-targeting antibodies, specifically anti-PD-1 antibodies, or agonistic OX40-specific antibodies, reduced tumour burden, prolonged survival, and induced tumour antigen-specific immunologic memory to prevent relapse in mouse tumour models. In addition, CPMV-based in situ vaccination combined with systemic low-dose CPA chemotherapy achieved impressive synergistic efficacy against 4T1 tumours. Low doses of CPA induce pro-immunogenic activity in tumour cells, including the hallmarks of immunogenic cell death (ICD). Activated APCs with CPMV ISV induce IL-12, IFN-, and IFN-and potentiated to induce an adaptive immune response. Data indicated that the combination of RT + CPMV enhanced efficacy over RT alone, and that this may be attributed to an expansion of T cells within the tumours. Many investigations examined the combination of PVNP in situ vaccination with chemotherapy, radiation therapy, checkpoint immunotherapies. Overall, multifunctional PVNPs can combine multiple treatment modalities into a single platform with ISV.

Conclusions

The development of plant viruses as expression vectors for pharmaceutical production has played an integral role in the emergence of plants as inexpensive and facile systems for the generation of therapeutic proteins. More recently, plant viruses have been designed as non-toxic nanoparticles which can target a variety of cancers via loading conventional therapeutic agents, tumour antigens, and immunotherapeutic agents. The tendency of PVNPs to interact with and become phagocytosed by innate immune cells can empower the immune system to slow or even reverse tumour progression.

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