Fluorescent bioinspired albumin/polydopamine nanoparticles and their interactions with Escherichia coli cells

Abstract

Inspired by the eumelanin aggregates in human skin, polydopamine nanoparticles (PDA NPs) are promising nanovectors for biomedical applications, especially because of their biocompatibility. We synthesized and characterized fluorescent PDA NPs of 10-25 nm diameter based on a protein containing a lysine-glutamate diad (bovine serum albumin, BSA) and determined whether they can penetrate and accumulate in bacterial cells to serve as a marker or drug nanocarrier. Three fluorescent PDA NPs were designed to allow for tracking in three different wavelength ranges by oxidizing BSA/PDA NPs (Ox-BSA/PDA NPs) or labelling with fluorescein 5-isothiocyanate (FITC-BSA/PDA NPs) or rhodamine B isothiocyanate (RhBITC-BSA/PDA NPs). FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs penetrated and accumulated in both cell wall and inner compartments of Escherichia coli (E. coli) cells. The fluorescence signals were diffuse or displayed aggregate-like patterns with both labelled NPs and free dyes. RhBITC-BSA/PDA NPs led to the most intense fluorescence in cells. Penetration and accumulation of NPs was not accompanied by a bactericidal or inhibitory effect of growth as demonstrated with the Gram-negative E. coli species and confirmed with a Gram-positive bacterial species (Staphylococcus aureus). Altogether, these results allow us to envisage the use of labelled BSA/PDA NPs to track bacteria and carry drugs in the core of bacterial cells.

Keywords: Escherichia coli; accumulation; albumin; antibacterial; fluorescence; nanoparticles; penetration; polydopamine.

Figure 1

Molecules of (a) lysine (K) and (b) glutamate (E). (c) Interactions between dopamine and KE sequence. (d) Possible structures of protein (grey)/polydopamine (black) composite particles (inspired from [14]). (e) Possible locations of organic nanoparticles in bacterial cells. Figure 1c was adapted with permission from [13]. Copyright 2018 American Chemical Society. This content is not subject to CC BY 4.0.

Figure 2

(a) Photographic image of a dopamine solution. Main reactions leading to an auto-polymerization of dopamine to (b) polydopamine or (c) to polydopamine nanoparticles in the presence of BSA (inspired from [13]). (d) Synthesis principle and photographic images of suspensions of fluorescent Ox-BSA/PDA NPs obtained by oxidation of pristine BSA/PDA NPs. (e) Synthesis principles and photographic images of fluorescent RhBITC-BSA/PDA NPs and FITC-BSA/PDA NPs obtained by using RhBITC- and FITC-labelled BSA, respectively.

Figure 3

Solutions and sizes of pristine and fluorescent BSA/PDA NPs. (a) Evidence of the inhibition of a PDA film deposition on the wall of the reaction beaker of (left) BSA/PDA solution in Tris buffer compared to (right) PDA solution in Tris buffer. (b) Mean hydrodynamic diameter of pristine BSA/PDA NPs as a function of the BSA/DA ratio. (c) Photograph of pristine BSA/PDA NPs after two years of storage. (d) Mean hydrodynamic diameter in number of pristine PDA/BSA-NPs depending on the solvent. (e) Mean hydrodynamic diameter in number of pristine PDA/BSA-NPs depending on the pH value. (f) Mean hydrodynamic diameter in number of pristine BSA/PDA NPs, Ox-BSA/PDA NPs, FITC-BSA/PDA NPs, and RhBITC-BSA/PDA NPs (all synthesized with a BSA/DA ratio of 10).

Figure 4

Absorption and emission spectra of pristine BSA/PDA NPs, Ox-BSA/PDA NPs, FITC-BSA/PDA NPs, and RhBITC-BSA/PDA NPs. (a) Absorption of pristine BSA/PDA NPs before and after oxidation. (b) Normalized absorption and fluorescence emission (excitation wavelength λ_exc = 375 nm) spectra of Ox-BSA/PDA NPs. (c) Normalized absorption and fluorescence emission (excitation wavelength λ_exc = 488 nm) spectra of FITC-BSA/PDA NPs. (d) Normalized absorption and fluorescence emission (excitation wavelength λ_exc = 550 nm) spectra of RhBITC-BSA/PDA NPs. Spectra were smoothed using the moving average method on three points.

Figure 5

Fluorescence characterization of E. coli cultures with FITC-BSA/PDA NPs, free FITC, RhBITC-BSA/PDA NPs, free RhBITC, or alone (NPs synthesized with a BSA/DA ratio of 10). (a) Typical fluorescence micrographs extracted from 3D-stack images measured with a high-resolution CLSM (Stellaris 5, Leica Biosystems, Wetzlar, Germany) (λ_exc of 405, 488, or 543 nm; range of λ_em of 415–482, 496–565, and 553–628 nm, respectively). (b) Maximal fluorescence intensity measured in bacterial cells for each condition (mean ± SD of all the micrographs of each condition). (c) Mean fluorescence intensity of bacterial cells for each condition (mean counts in bacterial cells per field) (mean ± SD of all complete bacteria in all micrographs of each condition).

Figure 6

Localization of the fluorescence emission related to E. coli cells. (a) Orthogonal views extracted from a 3D stack of a RhBITC-BSA/PDA NPs with E. coli sample measured with a high-resolution CLSM (Stellaris 5, Leica Biosystems, Wetzlar, Germany) (λ_exc of 405 and 543 nm; range of λ_em of 415–482 and 553–628 nm, respectively). (b) Longitudinal and transversal profiles of fluorescence intensity of a typical bacterial cell cultivated with RhBITC-BSA/PDA NPs (profiles are identified with numbers as displayed on the micrograph measured with the high-resolution Stellaris 5 CLSM as described in (a)).

Figure 7

Growth inhibition of E. coli populations with pristine BSA/PDA NPs from 0.2 to 2 mg/mL concentration or with antibiotics (solution of 10 µg/mL tetracycline and 0.1 µg/mL cefotaxime) (“T+C”; positive control) compared to E. coli culture without NPs (a), and with pristine and fluorescent Ox-BSA/PDA NPs and RhBITC-BSA/PDA NPs (1 mg/mL concentration) compared to E. coli culture without NPs (b). * and ** indicate significant differences to E. coli culture alone (p < 0.01 and p < 0.001, respectively).