Preparation of biocompatible heat-labile enterotoxin subunit B-bovine serum albumin nanoparticles for improving tumor-targeted drug delivery via heat-labile enterotoxin subunit B mediation

Abstract

Heat-labile enterotoxin subunit B (LTB) is a non-catalytic protein from a pentameric subunit of Escherichia coli. Based on its function of binding specifically to ganglioside GM1 on the surface of cells, a novel nanoparticle (NP) composed of a mixture of bovine serum albumin (BSA) and LTB was designed for targeted delivery of 5-fluorouracil to tumor cells. BSA-LTB NPs were characterized by determination of their particle size, polydispersity, morphology, drug encapsulation efficiency, and drug release behavior in vitro. The internalization of fluorescein isothiocyanate-labeled BSA-LTB NPs into cells was observed using fluorescent imaging. Results showed that BSA-LTB NPs presented a narrow size distribution with an average hydrodynamic diameter of approximately 254±19 nm and a mean zeta potential of approximately -19.95±0.94 mV. In addition, approximately 80.1% of drug was encapsulated in NPs and released in the biphasic pattern. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that BSA-LTB NPs exhibited higher cytotoxic activity than non-targeted NPs (BSA NPs) in SMMC-7721 cells. Fluorescent imaging results proved that, compared with BSA NPs, BSA-LTB NPs could greatly enhance cellular uptake. Hence, the results indicate that BSA-LTB NPs could be a potential nanocarrier to improve targeted delivery of 5-fluorouracil to tumor cells via mediation of LTB.

Keywords: 5-fluorouracil; bovine serum albumin; heat-labile enterotoxin subunit B; nanoparticle.

Figure 1

Schematic formation of 5-FU-loaded BSA-LTB nanoparticles. Abbreviations: 5-FU, 5-fluorouracil; BSA, bovine serum albumin; LTB, heat-labile enterotoxin subunit B.

Figure 2

Fluorescence microscope images of identification of LTB with immunofluorescence. Notes: (A) BSA-LTB nanoparticles; (B) BSA nanoparticles. Abbreviations: BSA, bovine serum albumin; LTB, heat-labile enterotoxin subunit B.

Figure 3

Morphological shapes (A) and particle sizes (B) of 5-FU-loaded BSA-LTB nanoparticles. Abbreviations: 5-FU, 5-fluorouracil; BSA, bovine serum albumin; LTB, heat-labile enterotoxin subunit B.

Figure 4

In vitro release profile of BSA-LTB nanoparticles in phosphate-buffered saline (pH 5.4 and pH 7.4 at 37°C) for 48 hours. Note: Data are expressed as the mean ± standard deviation (n=3). Abbreviations: BSA, bovine serum albumin; LTB, heat-labile enterotoxin subunit B.

Figure 5

In vitro uptake ability of NPs. Notes: Fluorescent microscopy analysis of the uptake of FITC-labeled BSA NPs and FITC-labeled BSA-LTB NPs in SMMC-7721 cells. The scale bar is 50 μm (A). Fluorescence spectrum analysis of the uptake of FITC-labeled BSA NPs and FITC-labeled BSA-LTB NPs in SMMC-7721 cells (n=3, *P<0.05, versus the corresponding BSA NPs) (B). Fluorescence spectrum analysis of the uptake of NPs labeled by FITC incubated with SMMC-7721 cells for 6 hours in media containing different amounts of free LTB (n=3, *P<0.05, versus the corresponding NP groups in media containing 5 μg/mL of LTB) (C). Abbreviations: BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; LTB, heat-labile enterotoxin subunit B; NP, nanoparticle.

Figure 6

Viability of cells after incubation with either free 5-FU, 5-FU-loaded BSANPs, or 5-FU-loaded BSA-LTB NPs, at various 5-FU concentrations (3.75–30 μg/mL). Notes: (A) After 24 hours; (B) after 48 hours; (C) after 72 hours (n=3) Abbreviations: 5-FU, 5-fluorouracil; BSA, bovine serum albumin; LTB, heat-labile enterotoxin subunit B; NPs, nanoparticles.

Figure 7

Cell apoptosis determined by Annexin V-FITC staining. Notes: Flow cytometer analysis of the apoptotic and necrotic cells (Q1: necrotic; Q2: late apoptotic; Q3: live; Q4: early apoptotic) after 48 hours of incubation with the free 5-FU, 5-FU-loaded BSA NPs, and 5-FU-loaded BSA-LTB NPs, respectively. Results are expressed as means ± standard deviation (n=3). Abbreviations: 5-FU, 5-fluorouracil; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; LTB, heat-labile enterotoxin subunit B; NPs, nanoparticles; PI, propidium iodide.