An efficient system for intracellular delivery of beta-lactam antibiotics to overcome bacterial resistance

Abstract

The "Golden era" of antibiotics is definitely an old story and this is especially true for intracellular bacterial infections. The poor intracellular bioavailability of antibiotics reduces the efficency of many treatments and thereby promotes resistances. Therefore, the development of nanodevices coupled with antibiotics that are capable of targeting and releasing the drug into the infected-cells appears to be a promising solution to circumvent these complications. Here, we took advantage of two natural terpenes (farnesyl and geranyl) to design nanodevices for an efficient intracellular delivery of penicillin G. The covalent linkage between the terpene moieties and the antibiotic leads to formation of prodrugs that self-assemble to form nanoparticles with a high drug payload between 55-63%. Futhermore, the addition of an environmentally-sensitive bond between the antibiotic and the terpene led to an efficient antibacterial activity against the intracellular pathogen Staphylococcus aureus with reduced intracellular replication of about 99.9% compared to untreated infected cells. Using HPLC analysis, we demonstrated and quantified the intracellular release of PenG when this sensitive-bond (SB) was present on the prodrug, showing the success of this technology to deliver antibiotics directly into cells.

Figure 1. (A) Chemical structure of the different moieties: Penicillin G sodium salt (PenG), Geraniol and E,E-Farnesol.

(B) Chemical structures of the GePenG (1), FaPenG (2) biocongugates. (C) Chemical structures of the bioconjugates harbouring an environmentally-sensitive bond between the terpene fraction and the penicillin G: GePenG-SB (3)and FaPenG-SB (4).

Figure 2. Cryogenic transmission electron microscopy images of FaPenG (A), FaPenG-SB (B), GePenG (C) and GePenG-SB (D) nanoparticles.

Scale bars = 100 nm.

Figure 3. Cell uptake of NPs by RAW 264.7 cells.

(A) Kinetics of uptake of fluorescently labelled NPs (FaPenG::BC and GePenG-SB::BC) and free probe (BC alone) by flow cytometry. (B) Confocal microscopy of RAW 264.7 cells treated (5 h and 24 h) with fluorescent NPs (FaPenG::BC and GePenG-SB::BC). NPs were fluorescently labelled in green with 0.5% BODIPY® FL C12 cholesteryl ester. Scale bars = 20 μm.

Figure 4. Intracellular survival of Staphylococcus aureus in RAW 264.7 cells after NPs treatment.

(A) Infected RAW 264.7 macrophages were incubated (20 h) or not with PenG, FaPenG NPs or GePenG-SB NPs at 20 μg.mL⁻¹, equiv. PenG. Control was untreated infected-macrophages. (B) Confocal microscopy of viable/dead intracellular bacteria (S. aureus) in RAW264.7 cells after 6 h treatment with PenG (B), FaPenG NPs (C) or GePenG-SB NPs (D) at 20 μg.mL⁻¹, equiv. PenG. Control was untreated infected-macrophages (A). After gentamicin treatment, cells were stained with LIVE/DEAD BacLight® kit. Living intracellular bacteria are labelled in green. Dead intracellular bacteria are labelled in red. Scale bars = 20 μm.

Figure 5. Quantification of intracellular Penicillin G in RAW 264.7 cells.

(A) After 90 min incubation with free PenG, FaPenG NPs or GePenGpH NPs (at 20 μg.mL⁻¹, equiv. PenG), intracellular PenG was estimated using HPLC method; n.d. means: no PenG detected. (B) After 2 h, 4 h and 6 h incubation with GePenG-SB NPs (20 μg.mL⁻¹, equiv. PenG), in the presence or in the absence of Probenecid (15 mM). Intracellular PenG was estimated using HPLC method.

Figure 6. In vitro release of PenG from NPs (FaPenG; FaPenG-SB; GePenG; GePenG-SB).

NPs (0.1 mg.mL⁻¹ equiv. PenG)were incubated at pH 4.5 or 7.4, in the presence or in the absence of cell lysates at 37 °C and PenG release was estimated by HPLC method.