Evaluation of antibacterial mechanism of polymeric nano‐particles by scanning transmission electron microscopy‐cathodoluminescence technique

Abstract

Introduction: Biofilm infection disease has become a serious problem as it is related to various types of refractory infections. Biofilms, with a three‐dimensional (3D) structure, are formed by bacterial cells covered by a thick film of extracellular polymeric substance matrix. This 3D matrix‐like structure can protect the bacterial cells within the biofilm from antibacterial drugs, rendering drug therapy against biofilm infection disease ineffective. This problem accelerates several refractory infections, such as periodontal disease and nosocomial infectious disease. Therefore, we have developed drug‐encapsulated polymeric nanoparticles (NPs) for drug delivery system to treat the biofilm infection disease. If microscopic observation of efficacy of prepared NPs at a nanoscale level can be carried out, preparation of suitable NPs for drug delivery can be determined. Therefore, the goal of this study is developing a scanning transmission electron microscopy‐cathodoluminescence (STEM‐CL) technique for evaluation of the antibacterial activity derived from NPs on the bacterial cells within the biofilm. Materials and method: S. epidermidis was used as a model biofilm forming bacterial strain. An ionic liquid (IL) 1‐butyl‐3‐methylimidazolium tetrafluoroborate was employed in sample preparation of STEM‐CL observation. The STEM‐CL observation was carried out by a JEM 2100M using a Gatan Vulcan TEM‐CL holder with a liquid nitrogen cooling system. For the TEM observation, the sample preparation method using IL and TEM observation using the cooling holder was combined. Samples for STEM and TEM observations were cooled down to 100 K. In addition, field emission‐scanning electron microscopy observation was also performed using a JXA‐8530FA. Using the LIVE/DEAD BacLight bacterial viability kit, antibacterial assays of the prepared NPs on the biofilm were performed. Polymeric NPs were prepared with polymeric poly (DL‐lactide‐co‐glycolide) (lactide:glycolide = 75:25, PLGA) NPs and Soluplus ® (Sol) micelles using the emulsion solvent diffusion (ESD) method. Clarithromycin and chitosan were used for antibacterial drug and surface modifier for NPs. The particle size and zeta potential of prepared NPs were determined using a Zetasizer Nano ZS90. Results and discussion: We developed the STEM‐CL technique for design of polymeric NPs on the biofilm. By CL imaging of fluorescence substance derived from NPs, the antibacterial activity and ability of the biofilm removal of the prepared NPs that have never been revealed were visualized at a nanoscale level. Moreover, a combination of several types of electron microscopy observations enables to reveal different antibacterial mechanism of respectively PLGA NPs and Sol micelle NPs. CS‐modified Sol micelle NPs can intrude into the bacterial cells within a short time of the treatment and exert high antibacterial activity by the induction of abnormal cell division and inhibition of cell division. The results of antibacterial assay using the LIVE/DEAD BacLight bacterial viability kit supported the electron microscopy studies. Based on the information obtained from the electron microscopy measurements, NPs can be suitably designed for the inhibition of biofilm formation and treatment against biofilm infections. The developed methodology can contribute to the nanoscale visualization of the antibacterial activity and other deformable composite materials.We expect this technique to be applicable to various fields such as pharmacy, engineering, biology, and medical science.