Tumor Microenvironment Responsive Pepper Mild Mottle Virus-Based Nanotubes for Targeted Delivery and Controlled Release of Paclitaxel

Abstract

Plant virus nanoparticles (PVNPs) have been widely used for drug delivery, antibody development and medical imaging because of their good biodegradation and biocompatibility. Particles of pepper mild mottle virus (PMMoV) are elongated and may be useful as drug carriers because their shape favours long circulation, preferential distribution and increased cellular uptake. Moreover, its effective degradation in an acidic microenvironment enables a pH-responsive release of the encapsulated drug. In this study, genetic engineering techniques were used to form rod-shaped structures of nanoparticles (PMMoV) and folated-modified PMMoV nanotubes were prepared by polyethylene glycol (PEG) to provide targeted delivery of paclitaxel (PTX). FA@PMMoV@PTX nanotubes were designed to selectively target tumor cells and to release the encapsulated PTX in response to pH. Efficient cell uptake of FA@PMMoV@PTX nanotubes was observed when incubated with tumor cells, and FA@PMMoV@PTX nanotubes had superior cytotoxicity to free PTX, as reflected by cell survival and apoptosis. This system is a strong candidate for use in developing improved strategies for targeted treatment of tumors.

Keywords: folate; nanotubes; pH-responsive; paclitaxel; pepper mild mottle virus.

FIGURE 1

Symptoms caused by PMMoV in N. benthamiana infected with a full-length cDNA infectious clone. (A) Phenotype of N. benthamiana plants agroinfiltrated with viral infectious clone combinations or empty agrobacterium (CK) at 28 days post infiltration. (B) Virions purified from leaves infected by the PMMoV infectious clones and observed by TEM. Bars represent 100 nm. (C) Detection of PMMoV CP by Western blot (upper panels) and RT-PCR confirming the presence of viral RNAs (lower panels) in systemic leaves of inoculated plants. Rubisco and Nb-UBC are the respective loading controls.

FIGURE 2

Characterization of FA@PMMoV@PTX nanotubes. (A) TEM images of FA@PMMoV@PTX nanotubes after incubation in media at different pH values. (B) The in vitro drug release of FA@PMMoV@PTX nanotubes in pH 7.4 medium, using free PTX as a control. (C) The in vitro drug release of FA@PMMoV@PTX nanotubes in media at pH 7.4, 5.0, and 1.0. (D) Images showing the fluorescence of FA@PMMoV@NR nanotubes after incubation in media at different pH values. (E) Images showing the fluorescence of FA@PMMoV@Dir nanotubes in MCF-7 cells and HUVEC (controls).

FIGURE 3

Uptake of FA@PMMoV nanotubes by tumor cells. (A) Images showing the fluorescence of FA@PMMoV@Dir nanotubes in MCF-7 cells, using PMMoV@Dir nanotubes as control and free FA as receptor competitor. (B) The semi-quantitative fluorescence signals from (A). (C) The fluorescence intensity of FA@PMMoV@Dir nanotubes in MCF-7 cells treated with different concentrations of free FA (0.1, 0.5, and 1.0 mg/ml).

FIGURE 4

The in vitro anti-tumor activity of FA@PMMoV@Dir nanotubes. (A) Cell viability of MCF-7 treated with different concentrations of FA@PMMoV@Dir nanotubes. (B) Images showing the fluorescence of MCF-7 cells co-stained with calcein-AM and Ethd-1 and treated with FA@PMMoV@Dir nanotubes. (C) The semi-quantitative values of fluorescence signals from (B). (D) Flow-cytometry-based apoptosis assay for MCF-7 cells treated with FA@PMMoV@Dir nanotubes.

SCHEME 1

FA@PMMoV@PTX nanotubes were prepared by PEG coprecipitation. FA@PMMoV@PTX nanotubes were taken up well by tumor cells because of the folate receptors overexpressed in them. A pH-responsive drug release in the acid tumor microenvironment led to good delivery of PTX.