Oligonucleotide Solid Nucleolipid Nanoparticles against Antibiotic Resistance of ESBL-Producing Bacteria

Abstract

Antibiotic resistance has become a major issue in the global healthcare system, notably in the case of Gram-negative bacteria. Recent advances in technology with oligonucleotides have an enormous potential for tackling this problem, providing their efficient intrabacterial delivery. The current work aimed to apply this strategy by using a novel nanoformulation consisting of DOTAU, a nucleolipid carrier, in an attempt to simultaneously deliver antibiotic and anti-resistance oligonucleotides. Ceftriaxone, a third-generation cephalosporin, was formulated with DOTAU to form an ion pair, and was then nanoprecipitated. The obtained solid nanocapsules were characterized using FT-IR, XRD, HPLC, TEM and DLS techniques and further functionalized by the anti-resistance ONα sequence. To obtain an optimal anti-resistance activity and encapsulation yield, both the formulation protocol and the concentration of ONα were optimized. As a result, monodispersed negatively charged nanoparticles of CFX-DOTAU-ONα with a molar ratio of 10:24:1 were obtained. The minimum inhibitory concentration of these nanoparticles on the resistant Escherichia coli strain was significantly reduced (by 75%) in comparison with that of non-vectorized ONα. All aforementioned results reveal that our nanoformulation can be considered as an efficient and relevant strategy for oligonucleotide intrabacterial delivery in the fight against antibiotic resistance.

Keywords: CTX-M15 ß-lactamase; ESBL-producing E. coli; antibiotic resistance; nanocapsules; nucleic acids; nucleolipid; oligonucleotides; solid nanoparticles.

Figure 1

Chemical structure of CFX and nucleolipids (DOTAU and diC16dT) ionized in aqueous medium at neutral pH.

Figure 2

X-ray diffractogram of CFX (blue), DOTAU (red) and ion pair (green).

Figure 3

Optimized formulation protocol and HPLC analysis results (in blue).

Figure 4

TEM images of (a) CFX–DOTAU NP and (b) Onα₆₀₀ NP.

Figure 5

Evolution of the effects of ONα NP size and zeta potential on ONα concentration during formulation (** p < 0.01, Student t-test).

Figure 6

Impact of incubation time on ONα₆₀₀ NP size, PDI and zeta potential, as compared to non-incubated CFX–DOTAU NP (at T0 time); * Student t-test was considered significant for p < 0.05.

Figure 7

Colloidal stability of CFX–DOTAU and ONα₆₀₀ NP characteristics (mean +/− SD): (left axis, expressed as % T0) size, zeta potential and (right axis) PDI.

Figure 8

CFX MIC of ONα NP on ESBL-producing E. coli compared to CFX–DOTAU NP and control conditions (* p < 0.05 and ** p < 0.01 Student t-test; NB: the concentration cited in brackets is the ONα incubation concentration during NP preparation).