Multi-walled carbon nanotubes increase antibody-producing B cells in mice immunized with a tetravalent vaccine candidate for dengue virus

Abstract

Background: In recent times, studies have demonstrated that carbon nanotubes are good candidates for use as vehicles for transfection of exogenous material into the cells. However, there are few studies evaluating the behavior of carbon nanotubes as DNA vectors and few of these studies have used multi-walled carbon nanotubes (MWCNTs) or carboxylated MWCNTs. Thus, this study aims to assess the MWCNTs' (carboxylated or not) efficiency in the increase in expression of the tetravalent vaccine candidate (TVC) plasmid vector for dengue virus in vitro using Vero cells, and in vivo, through the intramuscular route, to evaluate the immunological response profile.

Results: Multi-walled carbon nanotubes internalized by Vero cells, have been found in the cytoplasm and nucleus associated with the plasmid. However, it was not efficient to increase the messenger ribonucleic acid (mRNA) compared to the pure vaccine candidate associated with Lipofectamine(®) 2000. The in vivo experiments showed that the use of intramuscular injection of the TVC in combination with MWCNTs reduced the immune response compared to pure TVC, in a general way, although an increase was observed in the population of the antibody-producing B cells, as compared to pure TVC.

Conclusions: The results confirm the data found by other authors, which demonstrate the ability of nanotubes to penetrate target cells and reach both the cytoplasm and the cell nucleus. The cytotoxicity values are also in accordance with the literature, which range from 5 to 20 µg/mL. This has been found to be 10 µg/mL in this study. Although the expression levels are higher in cells that receive the pure TVC transfected using Lipofectamine(®) 2000, the nanotubes show an increase in B-cells producing antibodies.

Keywords: Dengue; Intramuscular route; Multi-walled carbon nanotubes; Vaccine; Vero cells.

Fig. 1

TEM of MWCNTs combined or not with plasmid DNA. TEM of MWCNT pure and combined with plasmid DNA. a MWCNT; b DNA-MWCNT; c c-MWCNT; d DNA- c-MWCNT. Scale bars in a indicate 200 nm; in b, c 1 μm and in d 500 nm

Fig. 2

TEM of Vero cells transfected with MWCNTs. TEM of Vero cells transfected with MWCNTs (arrows) (a) and (b) c-MWCNT (c) and (d). a, b and c, d show that they are in the cytoplasm and core, respectively. Details highlighted in the upper box. The scale bar indicates 2 μm in a (1 μm) and C (1 μm); 1 μm in b (1 μm) and d (500 nm). Details are indicated between parentheses. N core, C cytoplasm, M mitochondria

Fig. 3

RT-PCR of Vero cells transfected and MTT assay. a RT-PCR of Vero cells transfected with the empty pVAX plasmid, only TVC, MWCNT functionalized with TVC, and c-MWCNT functionalized with TVC. b, c MTT assay in Vero cells treated with c-MWCNTs pure preparations (c-MWCNT) (b) or functionalized with plasmid DNA (TVC-c-MWCNT) (c) to evaluate the cytotoxicity of preparations

Fig. 4

Western blot. Western blot and densitometry analysis of the different treatments. Were evaluated c-MWCNT and MWCNT functionalized with TVC, only TVC and TVC with Lipofectamine^® (TVC-LF) and empty pVAX. Densitometric analysis was performed using ImageJ software

Fig. 5

Immunophenotyping. Immunophenotyping of lymphocytes of animals subjected to four different treatments (pVAX, TVC, c-MWCNT, TVC-c-MWCNT). a B cells, b T CD4+ cells, c T CD8+ cells, d CD62L low cells

Fig. 6

Lymphoproliferation and cytokine production. a Lymphoproliferation of lymphocytes of animals subjected to four different treatments (TVC, c-MWCNT, TVC-c-MWCNT) when stimulated with the four serotypes of dengue virus, the medium (negative control), and Concanavalin A (ConA) (positive control). b–d Featured cytokine that had a significant increase, showing the levels produced by lymphocytes of animals subjected to four different treatments (TVC, c-MWCNT, TVC-c-MWCNT) when stimulated with the four serotypes of the dengue virus and the medium (negative control)