Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells

Abstract

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Keywords: carbon nanoparticles; drug delivery; internalization; melittin.

Figure 1

Size distribution of MEL, GN, GO, and complexes. (A)—MEL; (B)—GN; (C)—GO; (D)—MGN; (E)—MGO.

Figure 2

Zeta potential of MEL, GN, GO, and complexes. (A)—MEL; (B)—GN; (C)—GO; (D)—MGN; (E)—MGO.

Figure 3

FT-IR spectra. (A)—GN, MEL, MGN; (B)—GO, MEL, MGO.

Figure 4

Immunofluorescence images of MCF-7 line cells stained for actin filaments (red) and nuclei (blue). (A)—control, (B)—MEL-treated group, (C)—GN-treated group, (D)—GO-treated group, (E)—MGN-treated group, and (F)—MGO-treated group.

Figure 5

Immunofluorescence images of HFFF2 line cells stained for actin filaments (red) and nuclei (blue). (A)—control, (B)—MEL-treated group, (C)—GN-treated group, (D)—GO-treated group, (E)—MGN-treated group, and (F)—MGO-treated group.

Figure 6

Intracellular pH in cell lines after treatment with MEL, nanoparticles, and complexes. C signifies the control samples and the results are mean values ± standard deviation. “*” indicates statistically significant differences in comparison to the control (p-value ≤ 0.05).

Figure 7

Potential of cell membrane in cell lines after treatment with MEL, nanoparticles, and complexes. C signifies the control samples and the results are mean values ± standard deviation. “*” indicates statistically significant differences in comparison to the control (p-value ≤ 0.05).

Figure 8

Effect of transport inhibitors on the internalization of M, GN, GO, MGN, and MGO into MDA-MB-231 cells. C signifies the control samples and the results are mean values ± standard deviation. “*” indicates statistically significant differences in comparison to the control (p-value ≤ 0.05).

Figure 9

Effect of transport inhibitors on the internalization of M, GN, GO, MGN, and MGO into MCF-7 cells. C signifies the control samples and the results are mean values ± standard deviation. “*” indicates statistically significant differences in comparison to the control (p-value ≤ 0.05).

Figure 10

Effect of transport inhibitors on the internalization of M, GN, GO, MGN, and MGO into HFFF2 cells. C signifies the control samples and the results are mean values ± standard deviation. “*” indicates statistically significant differences in comparison to the control (p-value ≤ 0.05).

Figure 11

Antibody array analysis of human cell membrane receptor (original drafts) in MDA-MB-231 cells. (A)—control group, (B)—MEL-treated group, (C)—MGN-treated group, and (D)—MGO-treated group. Results were normalized and compared to a dots control sample.

Figure 12

Antibody array analysis of human cell membrane receptor (original drafts) in MCF-7 cells. (A)—control group, (B)—MEL-treated group, (C)—MGN-treated group, and (D)—MGO-treated group. Results were normalized and compared to a dots control sample.

Figure 13

Scheme of MGN and MGO complexes forming.

Figure 14

Mechanism of action of MEL, MGN, and MGO on MDA-MB-231 and MCF-7 cells; CDE—caveolin-dependent endocytosis; CME—clathrin-mediated endocytosis, MDT—microtubule-dependent transport, DDE—dynamin-dependent endocytosis.