Amine/thiol-reactive chemistries are commonly used to conjugate antibodies to pharmaceuticals or nanoparticles. Yet, these conjugation strategies often result in unfavorable outcomes such as heterogeneous antibody display with hindered biological activity or aggregation due to multivalent interactions of the antibody and nanoparticles. Here, we report the application of a site-specific and enzymatically driven antibody conjugation strategy to functionalize virus-based nanoparticles (VNPs). Specifically, an azide-handle was introduced into the Fc region of a set of immunoglobulins using a two-step enzymatic reaction: (1) cleavage of N-linked glycan in the Fc region by a glycosidase and (2) conjugation of a chemically reactive linker (containing an azide functional handle) using a microbial transglutaminase. Conjugation of the azide-functional antibodies to several VNPs was achieved by making use of strain-promoted azide–alkyne cycloaddition. We report the conjugation of three immunoglobulin (IgG) isotypes (human IgG from sera, anti-CD47 Rat IgG2a, κ, and Trastuzumab recombinant humanized IgG1, κ) to the plant virus cowpea mosaic virus (CPMV) and the lysine mutant of tobacco mosaic virus (TMVlys) as well as bacteriophage Qβ. Site-specific conjugation resulted in stable and functional antibody-VNP conjugates. In stark contrast, the use of heterobifunctional linkers targeting thiols and amines on the antibodies and VNPs, respectively, led to aggregation due to nonspecific and multivalent coupling between the antibodies and VNPs. We demonstrate that antibody-VNP conjugates were functional, and Trastuzumab-displaying VNPs targeted HER2-positive SKOV-3 human ovarian cancer cells. This bioconjugation strategy adds to the portfolio of methods that can be used for designing functional antibody-VNP conjugates.

Prognosis and treatment of metastatic cancer continues to be one of the most difficult and challenging areas of oncology. Treatment usually consists of chemotherapeutics, which may be ineffective due to drug resistance, adverse effects, and dose‐limiting toxicity. Therefore, novel approaches such as immunotherapy have been investigated to improve patient outcomes and minimize side effects. S100A9 is a calcium‐binding protein implicated in tumor metastasis, progression, and aggressiveness that modulates the tumor microenvironment into an immunosuppressive state. S100A9 is expressed in and secreted by immune cells in the pre‐metastatic niche, as well as, post‐tumor development, therefore making it a suitable targeted for prophylaxis and therapy. In previous work, it is demonstrated that cowpea mosaic virus (CPMV) acts as an adjuvant when administered intratumorally. Here, it is demonstrated that systemically administered, S100A9‐targeted CPMV homes to the lungs leading to recruitment of innate immune cells. This approach is efficacious both prophylactically and therapeutically against lung metastasis from melanoma and breast cancer. The current research will facilitate and accelerate the development of next‐generation targeted immunotherapies administered as prophylaxis, that is, after surgery of a primary breast tumor to prevent outgrowth of metastasis, as well as, therapy to treat established metastatic disease.

Cardiovascular thrombotic disease is an underlying cause of stroke, myocardial infarction and pulmonary embolism - some of the leading causes of death worldwide. Reperfusion therapy with anticoagulant, antiplatelet, and fibrinolytic agents has significantly reduced early mortality and morbidity from acute myocardial infarction and stroke. Nevertheless, bleeding side effects (e.g., intracranial hemorrhage) associated with the anti-thrombotic therapy can offset its benefits and limit its applicability to strictly defined short therapeutic windows. We have previously shown that elongated plant virus based nanoparticles can target cardiovascular thrombi and exhibited their utility for the delivery of streptokinase in an ex vivo model of thrombosis. Herein, we build upon our previous findings and demonstrate plant viral delivery of the current standard-of-care tissue plasminogen activator (tPA). Studies on a pre-clinical mouse model of arterial thrombosis indicate that while the therapeutic efficacy of free tPA and tPA-conjugated TMV are similar, the safety profile of the tPA-TMV formulation is improved, i.e. administration of the latter has less impact on hemostasis as demonstrated by decreased bleeding time. Thus, our data suggest that TMV-based delivery of thrombolytic therapies could be a promising and safer alternative to reperfusion therapy with the tPA.

The development of plant viral nanoparticles (VNP) loaded with different molecular versions of a photodynamic drug is described. Specifically, tobacco mosaic virus (TMV) and tobacco mild green mosaic virus (TMGMV) are developed as drug carriers that encapsulate the monocationic, dicationic, tricationic, and tetracationic versions of a porphyrin‐based photosensitizer drug (Zn‐Por). While TMV has been extensively explored for various nanotechnology applications, this is the first study investigating TMGMV for medical applications. Light‐activated cancer cell killing of Zn‐Por‐loaded VNPs is studied in vitro using melanoma and cervical cancer models. Native and nucleolin‐targeted VNP drug carriers are developed and their efficacy assessed. A fivefold increase in cancer cell killing is observed using nucleolin‐targeted TMV loaded with tricationic Zn‐Por and displaying the nucleolin‐specific F3 peptide.

Thromboembolic conditions are a leading cause of death worldwide, and deep vein thrombosis (DVT), or occlusive venous clot formation, is a critical and rising problem that contributes to damage of vital organs, long-term complications, and life-threatening conditions such as pulmonary embolism. Early diagnosis and treatment are correlated to better prognosis. However, current technologies in these areas, such as ultrasonography for diagnostics and anticoagulants for treatment, are limited in terms of their accuracy and therapeutic windows. In this work, we investigated targeting myeloid related protein 14 (MRP-14, also known as S100A9) using plant virus-based nanoparticle carriers as a means to achieve tissue specificity aiding prognosis and therapeutic intervention. We used a combinatorial peptide library screen to identify peptide ligands that bind MRP-14. Candidates were selected and formulated as nanoparticles by using cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV). Intravascular delivery of our MRP-14-targeted nanoparticles in a murine model of DVT resulted in enhanced accumulation in the thrombi and reduced thrombus size, suggesting application of nanoparticles for molecular targeting of MRP-14 could be a promising direction for improving DVT diagnostics, therapeutics, and therefore prognosis.

Stable suspensions of magnetic nanoparticles (MNPs) with large magnetic moment, m, per particle have tremendous utility in a wide range of biological applications. However, due to the strong magnetic coupling interactions often present in these systems, it is challenging to stabilize individual, high moment, ferro- and ferrimagnetic nanoparticles. A novel approach to encapsulate large, i.e., >100 nm, ferrimagnetic zinc ferrite nanocubes (ZFNCs) with silica after an intermediary layer-by-layer polyelectrolyte deposition step is described in this paper. The seed ZFNCs are uniform in shape and size and have high saturation mass magnetic moment (σs ~100 emu/g, m~4×10−13 emu/particle at 150 Oe). For the MNP system described within, successful silica encapsulation and creation of discrete ZFNCs were realized only after depositing polyelectrolyte multilayers composed of alternating polyallylamine and polystyrene sulfonate. Without the intermediary polyelectrolyte layers, magnetic dipole-dipole interactions led to the formation of linearly chained ZFNCs embedded in a silica matrix. Characterization of particle samples was performed by electron microscopy, energy-dispersive X-ray spectroscopy, infrared spectroscopy, powder X-ray diffraction, dynamic light scattering (hydrodynamic size and ζ-potential), and vibrating sample magnetometry. The results of these characterizations, which were performed after each of the synthetic steps, and synthetic details are presented.

TMV-based plant virus nanoparticles targeting S100A9 exhibited high specificity to atherosclerotic lesions in ApoE−/− mice, which provides a new diagnostic strategy for atherosclerosis with high risk factors.

This review examines the advantages and limitations of in vitro health care diagnostic tests that utilize nanoparticles (e.g. noble metal, quantum dot, and magnetic). It includes a brief overview of their unique properties, syntheses, and applicable readout strategies. This is followed by a brief synopsis of the obstacles faced when attempting to translate nanoparticle-based diagnostics from the R&D laboratory to the clinic and other arenas (i.e. the difficulties common to in vitro diagnostics), and then by a much more in-depth examination of the need to control and characterize a range of nanoparticle properties (e.g. size, shape, surface composition, and stability) when making this transition. Expert commentary: The review wraps up with a short commentary and perspective for the next five years, focusing on possible guidelines for nanoparticle characterization.