Featured Article: Delivery of chemotherapeutic vcMMAE using tobacco mosaic virus nanoparticles

Abstract

The first-line treatment for non-Hodgkin's lymphoma is chemotherapy. While generally well tolerated, off-target effects and chemotherapy-associated complications are still of concern. To overcome the challenges associated with systemic chemotherapy, we developed a biology-inspired, nanoparticle drug delivery system (nanoDDS) making use of the nucleoprotein components of the tobacco mosaic virus (TMV). Virus-based nanoparticles, including the high-aspect ratio soft nanorods formed by TMV, are growing in popularity as nanoDDS due to their simple genetic and chemical engineerability, size and shape tunability, and biocompatibility. In this study, we used bioconjugation to modify TMV as a multivalent carrier for delivery of the antimitotic drug valine-citrulline monomethyl auristatin E (vcMMAE) targeting non-Hodgkin's lymphoma. We demonstrate successful synthesis of the TMV-vcMMAE; data indicate that the TMV-vcMMAE particles remained structurally sound with all of the 2130 identical TMV coat proteins modified to carry the therapeutic payload vcMMAE. Cell uptake using Karpas 299 cells was confirmed with TMV particles trafficking to the endolysosomal compartment, likely allowing for protease-mediated cleavage of the valine-citrulline linker for the release of the active monomethyl auristatin E component. Indeed, effective cell killing of non-Hodgkin's lymphoma in vitro was demonstrated; TMV-vcMMAE was shown to exhibit an IC₅₀ of ∼250 nM. This study contributes to the development of viral nanoDDS. Impact statement Due to side effects associated with systemic chemotherapy, there is an urgent need for the development of novel drug delivery systems. We focus on the high-aspect ratio nanotubes formed by tobacco mosaic virus (TMV) to deliver antimitotic drugs targeted to non-Hodgkin's lymphoma. Many synthetic and biologic nanocarriers are in the development pipeline; the majority of systems are spherical in shape. This may not be optimal, because high-aspect ratio filaments exhibit enhanced tumor homing, increased target cell interactions and decreased immune cell uptake, and therefore have favorable properties for drug delivery compared to their spherical counterparts. Nevertheless, the synthesis of high-aspect ratio materials at the nanoscale remains challenging; therefore, we turned toward the nucleoprotein components of TMV as a biologic nanodrug delivery system. This work presents groundwork for the development of plant virus-based vehicles for use in cancer treatment.

Keywords: Tobacco mosaic virus; chemotherapy; drug delivery; lymphoma; nanoparticle; valine-citrulline monomethyl auristatin E.

Figure 1

Chemical structures and bioconjugation reaction. (a) Bioconjugation steps: TMV-lysine was first reacted with SATP via NHS chemistry, TMV-SATP’s thiol group was then deprotected using hydroxylamine, and finally TMV-SH was reacted with the maleimide group of vcMMAE, resulting in TMV-vcMMAE. (b) Chemical structure of vcMMAE. (A color version of this figure is available in the online journal.) vcMMAE: valine-citrulline monomethyl auristatin E.

Figure 2

Biochemical characterization of the vcMMAE-loaded TMV. (a) SDS-PAGE gel of TMV-lys conjugation process. Lane 1: TMV-lys, Lanes 2 and 4: TMV-SATP, and Lanes 3 and 5: TMV-vcMMAE. (b) Densitometry analysis was performed using band analysis tool and ImageJ software showing two distinct bands: CP vs. CP-vcMMAE. (c) TEM micrograph of negatively stained TMV-vcMMAE. (A color version of this figure is available in the online journal.) CP-vcMMAE: coat protein-valine-citrulline monomethyl auristatin E

Figure 3

Karpas 299 cell interactions with TMV. Nuclei were stained with DAPI (blue), endolysosomes were stained with mouse anti-human Lamp-1 antibody followed by secondary Alexa Fluor 488 goat anti-mouse antibody (green), and TMV particles were stained with primary rabbit anti-TMV antibody followed by secondary Alexa Fluor 555 goat anti-rabbit antibody (red). Merging of all three channels indicated co-localization of TMV with the endolysosomes (yellow). (A color version of this figure is available in the online journal.) DAPI: 4',6-diamidino-2-phenylindole; TMV: tobacco mosaic virus

Figure 4

Cell viability assay. Alamar Blue cell viability assay of Karpas 299 cells after 72 hours of treatment with vcMMAE (purple) and TMV-vcMMAE (blue) at 0.02 nM to 200 nM vcMMAE; non-conjugated TMV was also tested using the protein concentration corresponding to the highest vcMMAE dose (200 nM), toxicity of TMV was not observed. Error bars denote the standard deviation (experiments were done in triplicate). Data were analyzed and graphed using Prism® v6.0 b software. (A color version of this figure is available in the online journal.) vcMMAE: valine-citrulline monomethyl auristatin E; TMV: tobacco mosaic virus