Tobacco mosaic virus-based protein nanoparticles and nanorods for chemotherapy delivery targeting breast cancer

Abstract

Drug delivery systems are required for drug targeting to avoid adverse effects associated with chemotherapy treatment regimes. Our approach is focused on the study and development of plant virus-based materials as drug delivery systems; specifically, this work focuses on the tobacco mosaic virus (TMV). Native TMV forms a hollow, high aspect-ratio nanotube measuring 300×18nm with a 4nm-wide central channel. Heat-transformation can be applied to TMV yielding spherical nanoparticles (SNPs) measuring ~50nm in size. While bioconjugate chemistries have been established to modify the TMV rod, such methods have not yet been described for the SNP platform. In this work, we probed the reactivity of SNPs toward bioconjugate reactions targeting lysine, glutamine/aspartic acid, and cysteine residues. We demonstrate functionalization of SNPs using these chemistries yielding efficient payload conjugation. In addition to covalent labeling techniques, we developed encapsulation techniques, where the cargo is loaded into the SNP during heat-transition from rod-to-sphere. Finally, we developed TMV and SNP formulations loaded with the chemotherapeutic doxorubicin, and we demonstrate the application of TMV rods and spheres for chemotherapy delivery targeting breast cancer.

Keywords: Breast cancer; Drug delivery; Nanoparticles; Nanorods; Tobacco mosaic virus.

Fig. 1

Bioconjugate chemistries targeting lysine, cysteine, aspartic/glutamic acids (carboxylic acids) and tyrosine on TMV and SNP-based nanoparticle formulations (see Sections 2.1.1 and 2.1.2).

Fig. 2

A) Quantification of Cy5 attachment to SNP coat proteins (CPs) as a function of molar excess used. Averaged values from three independent experiments are shown error bars represent the standard deviation. Prism® 6.0b software was used to analyze and plot the data. B) SDS-PAGE of Cy5-labeled and native SNP and TMV visualized under UV light (to detect the Cy5 label) and under white light after Coomassie Blue (CB) staining (to detect the protein). Cy5-labeled SNPs after modification of amines (blue), thiols (green), and carboxylic acids (red) were analyzed where SNPs were probed with molar excess of 2, 5, 10, 20, 40 Cy5 per CP for amine- and thiol-selective reactions and 5, 10, 20, 40 Cy5 per CP targeting carboxylates.

Fig. 3

A) Schematic detailing the reactions leading to biotinylated TMV and SNP formulations, and B) corresponding TEM images of negative (UAc)-stained samples after immunogold staining using a gold-labeled anti-biotin antibody; the scale bar is 50 nm. C) Averaged diameter and standard deviation of the modified SNPs as determined by quantitative SEM image analysis.

Fig. 4

TMV– and SNP–cell interactions using MDA-MB-231 and MCF-7 cells; Cy5-labeled nanoparticle formulations (TMV shown in blue, SNP shown in red) were incubated with cells in medium for 6 h, then washed, fixed, and analyzed using BD LSR II or BD FACS Aria flow cytometer, and 10,000 gated events per sample were collected (all samples were analyzed in triplicates). Data were plotted and analyzed using FlowJo® 10.6 software; the mean fluorescence intensity (Cy5 MFI) or the percentage TMV/SNP-positive cells are plotted (error bars show the standard deviation).

Fig. 5

A) Cell viability of MDA-MB-231 and MCF-7 cells after treatment with DOX (black), _DOXTMV (blue), _DOXSNP (red), and _eDOXSNP (pink). Cell viability was determined after 72 h using MTT assay; dose-dependency was evaluated testing a concentration range of 0.1–10 μM normalized to DOX. Data shown are averaged data from three biological replicas (each performed in triplicates); error bars indicate the standard deviation. B) IC50 values and standard deviation comparing DOX, _DOXTMV, _DOXSNP, and _eDOXSNP. Data were analyzed and graphed using Prism® v6.0b software.