Background: After the golden age of antibiotic discovery, bacterial infections still represent a major challenge for public health worldwide. The biofilm mode of growth is mostly responsible for chronic infections that current therapeutics fail to cure and it is well-established that novel strategies must be investigated. Particulate drug delivery systems are considered as a promising strategy to face issues related to antibiotic treatments in a biofilm context. Particularly, poly-lactic acid (PLA) nanoparticles present a great interest due to their ability to migrate into biofilms thanks to their submicronic size. However, questions still remain unresolved about their mode of action in biofilms depending on their surface properties. In the current study, we have investigated the impact of their surface charge, firstly on their behavior within a bacterial biofilm, and secondly on the antibiotic delivery and the treatment efficacy. Results: Rifampicin-loaded PLA nanoparticles were synthetized by nanoprecipitation and characterized. A high and superficial loading of rifampicin, confirmed by an in silico simulation, enabled to deliver effective antibiotic doses with a two-phase release, appropriate for biofilm-associated treatments. These nanoparticles were functionalized with poly-l-lysine, a cationic peptide, by surface coating inducing charge reversal without altering the other physicochemical properties of these particles. Positively charged nanoparticles were able to interact stronger than negative ones with Staphylococcus aureus, under planktonic and biofilm modes of growth, leading to a slowed particle migration in the biofilm thickness and to an improved retention of these cationic particles in biofilms. While rifampicin was totally ineffective in biofilms after washing, the increased retention capacity of poly-l-lysine-coated rifampicin-loaded PLA nanoparticles has been associated with a better antibiotic efficacy than uncoated negatively charged ones. Conclusions: Correlating the carrier retention capacity in biofilms with the treatment efficacy, positively charged rifampicin-loaded PLA nanoparticles are therefore proposed as an adapted and promising approach to improve antibiotic delivery in S. aureus biofilms.

We describe a theoretical framework for understanding the heteronuclear version of the third spin assisted recoupling polarization transfer mechanism and demonstrate its potential for detecting long-distance intramolecular and intermolecular ¹⁵N–¹³C contacts in biomolecular systems. The pulse sequence, proton assisted insensitive nuclei cross polarization (PAIN-CP) relies on a cross term between ¹H–¹⁵N and ¹H–¹³C dipolar couplings to mediate zero- and/or double-quantum ¹⁵N–¹³C recoupling. In particular, using average Hamiltonian theory we derive effective Hamiltonians for PAIN-CP and show that the transfer is mediated by trilinear terms of the form N^±C^∓H_z (ZQ) or N^±C^±H_z (DQ) depending on the rf field strengths employed. We use analytical and numerical simulations to explain the structure of the PAIN-CP optimization maps and to delineate the appropriate matching conditions. We also detail the dependence of the PAIN-CP polarization transfer with respect to local molecular geometry and explain the observed reduction in dipolar truncation. In addition, we demonstrate the utility of PAIN-CP in structural studies with ¹⁵N–¹³C spectra of two uniformly ¹³C,¹⁵N labeled model microcrystalline proteins—GB1, a 56 amino acid peptide, and Crh, a 85 amino acid domain swapped dimer (MW = 2 × 10.4 kDa). The spectra acquired at high magic angle spinning frequencies (ω_r/2π > 20 kHz) and magnetic fields (ω_0H/2π = 700–900 MHz) using moderate rf fields, yield multiple long-distance intramonomer and intermonomer ¹⁵N–¹³C contacts. We use these distance restraints, in combination with the available x-ray structure as a homology model, to perform a calculation of the monomer subunit of the Crh protein.

β-TrCP is the F-box protein component of an Skp1/Cul1/F-box (SCF)-type ubiquitin ligase complex. Biochemical studies have suggested that β-TrCP targets the oncogenic protein β-catenin for ubiquitination and followed by proteasome degradation. To further elucidate the basis of this interaction, a complex between a 32-residue peptide from β-catenin containing the phosphorylated motif DpSGXXpS (P-β-Cat^17–48) and β-TrCP was studied using Saturation Transfer Difference (STD) Nuclear Magnetic Resonance (NMR) experiments. These experiments make it possible to identify the binding epitope of a ligand at atomic resolution. An analysis of STD spectra provided clear evidence that only a few of the 32 residues receive the largest saturation transfer. In particular, the amide protons of the residues in the phosphorylated motif appear to be in close contact to the amino acids of the β-TrCP binding pocket. The amide and aromatic protons of the His²⁴ and Trp²⁵ residues also receive a significant saturation transfer. These findings are in keeping with a recently published x-ray structure of a shorter β-catenin fragment with the β-TrCP1-Skp1 complex and with the earlier findings from mutagenesis and activity assays. To better characterize the ligand-protein interaction, the bound conformation of the phosphorylated β-catenin peptide was obtained using TRansfer Nuclear Overhauser Effect SpectroscopY (TRNOESY) experiments. Finally, we obtained the bound structure of the phosphorylated peptide showing the protons identified by STD NMR as exposed in close proximity to the molecule surface.