Cancer Theranostic Applications of Albumin-Coated Tobacco Mosaic Virus Nanoparticles

Abstract

Nanotechnology holds great promise in cancer drug delivery, and of particular interest are theranostic approaches in which drug delivery and imaging are integrated. In this work, we studied and developed the plant virus tobacco mosaic virus (TMV) as a platform nanotechnology for drug delivery and imaging. Specifically, a serum albumin (SA)-coated TMV formulation was produced. The SA coating fulfils two functions: SA provides a stealth coating for enhanced biocompatibility; it also acts as a targeting ligand enabling efficient tumor accumulation of SA-TMV versus TMV in mouse models of breast and prostate cancer. We demonstrate drug delivery of the chemotherapy doxorubicin (DOX); TMV-delivered DOX outperformed free DOX, resulting in significant delayed tumor growth and increased survival. Furthermore, we demonstrated the ability of SA-coated TMV loaded with chelated Gd(DOTA) for magnetic resonance imaging detection of tumors. In the future, we envision the application of such probes as theranostic, where first imaging is performed to assess whether the nanoparticles are effective at targeting a particular patient tumor. If targeting is confirmed, the therapeutic would be added and treatment can begin. The combination of imaging and therapy would allow to monitor disease progression and therefore inform about the effectiveness of the drug delivery approach.

Keywords: cancer nanotechnology; drug delivery; imaging; theranostics; tobacco mosaic virus.

Figure 1.

Bioconjugation of SA and cargo molecules to TMV. (A) schematic representation of conjugation of SA to external surface of TMV-Lys and conjugation of Cy5, Gd(DOTA), and DOX to internal Glu residues of TMV: (i) conjugation of N-hydroxysuccinimide (NHS)-ester of SAT-(PEG)₄ to external amine groups of SA; (ii) conjugation of SM-(PEG)₄ to TMV’s external Lys residues, followed by combining products (i + ii). (B) Schematic representation of conjugation of Cy5 or Gd(DOTA) and DOX to internal Glu residues of TMV: (i) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) conjugation of propargyl amine to glutamic acid; (ii) alkyne–azide cycloaddition of Cy5-azide/GD(DOTA)-azide to product of reaction (i); (iii) EDC conjugation of adipic acid dihydrazide (AAD) to Glu; (iv) formation of hydrazone bond between DOX and the product of reaction (iii). (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of TMV particles before and after conjugation. Successful conjugation of SA (M_w = 66 kDa; apparent M_w on the gel <64 kDa) to TMVcp (M_w = 17 kDa) in both SA-TMV-Cy5 and SA-TMV−/−DOX is indicated by the presence of multiple high molecular weight (>64 kDa) protein bands corresponding to SA-TMVcp conjugate (theoretical M_w of SA-TMVcp monomer is ~83 kDa). Free SA was used as a reference. (D) UV–vis spectra of TMV-Lys and its conjugates with Cy5, DOX, and SA. The spectra were normalized to A₂₆₀—maximum absorbance of TMV nucleic acid indicative of TMV concentration. Successful conjugation of Cy5 or DOX to internal cavity of TMV-Lys is indicated by the presence of absorbance peaks at 646 nm for Cy5 or 481 nm for DOX. (E) Transmission electron microscopy (TEM) images of SA-TMV-Cy5 (left) and SA-TMV−/−DOX (right) demonstrate structural integrity of TMV particles after the conjugation. Scale bars represent 500 nm.

Figure 2.

In vivo biodistribution of SA-TMV-Cy5 vs TMV-Cy5 in murine models of MDA-MB-231 and 4T1 cancer. (A) Representative Maestro fluorescence images of biodistribution of SA-TMV-Cy5 particles in heterotopic model of human MDA-MB231 in NCR nu/nu mice (10 h post-i.v. administration). (B) Maestro fluorescence images of biodistribution of SA-TMV-Cy5 particles in heterotopic model of murine 4T1 in Balb/c mice. (C,D) Quantification of particle accumulation per unit area of different organs from panels A and B, respectively (i.e., average fluorescence signal per image pixel). (E,F) Quantification of % total particle accumulation in different organs in panels A and B, respectively (i.e., total fluorescence signal in each organ normalized to total fluorescence of all organs). SA stealth coating significantly improves accumulation of TMV particles in tumor mass in comparison to “bare” TMV particles.

Figure 3.

In vitro assays of nanoparticle uptake and cytotoxicity. (A,B) Quantification of cellular uptake of DOX, TMV-DOX, and SA-TMV-DOX in vitro in isolated MDA-MB231 and 4T1 cells, respectively. SA stealth coating decreases cellular internalization of therapeutic SA-TMV-DOX particles. (C,D) 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) viability and proliferation assay using MDA-MB231 and 4T1 cells, respectively. TMV-DOX, TMV−/−DOX, and SA-coated versions thereof were tested; free DOX, TMV, and SA-TMV were used as controls.

Figure 4.

pH-driven cleavage of DOX from SA-TMV-DOX particles. (A) Schematic representation of cleavage of pH-sensitive hydrazone bond between DOX and TMV (C═N double bond) in acidic conditions. (B) Schematic representation of DOX being released from the internal cavity of TMV upon cleavage of hydrazone link. (C) Agarose gel analysis of SA-TMV−/−DOX samples after forced cleavage of DOX from TMV in acidic conditions (pH = 5 and pH = 6.5). The cleavage of DOX is indicated by increased fluorescence intensity in the top side of the gel proportional to incubation time in acidic conditions. Positively charged (free) DOX molecules separate from TMV (unable to enter the pores of agarose gel) and travel toward negatively charged electrode (i.e., cathode). Free DOX was run as a reference.

Figure 5.

In vivo efficacy of SA-TMV−/−DOX in MDA-MB231 mouse model. SA-TMV−/−DOX and free DOX were administered intravenously at a dose of 1 mg/kg every 3 days; a total of 10 treatments was given over a 4-week time course. One group of animals was treated with PBS. (A) Tumor growth curves, (B) survival plots.

Figure 6.

In vivo diagnostics of PC3 tumor in NCR nu/nu mice by MRI and biodistribution of TMV-based contrast agents. (A) The SA-TMV-Gd(DOTA) brightened the PC3 tumor at 6 h after injection as compared with PEG-TMV-Gd(DOTA) contrast agent; slow recovery of T₁ relaxation times occurred 24 h post-injection. (B) Quantitative normalized T₁ decreased ~25% for SA-TMV-Gd(DOTA) at 6 h after injection and ~9% for PEG-TMV-Gd(DOTA) contrast agent. (C) Biodistribution of SA-TMV-Gd(DOTA) and PEG-TMV-Gd(DOTA) particles in NCR nu/nu mice bearing PC-3 tumor.