Probing the size limit for nanomedicine penetration into Burkholderia multivorans and Pseudomonas aeruginosa biofilms

Encapsulation of antibiotics into nanoparticles is a potential strategy to eradicate biofilms. To allow further optimization of nanomedicines for biofilm eradication, the influence of the nanoparticle size on the penetration into dense biofilm clusters needs to be investigated. In the present study, the penetration of nanoparticles with diameters ranging from 40 to 550 nm into two biofilms, Burkholderia multivorans LMG 18825 and Pseudomonas aeruginosa LMG 27622, was evaluated using confocal microscopy. Through image analysis, the percentage of particles able to penetrate into dense biofilm clusters was calculated. The size cut off for optimal penetration into biofilm clusters was located around 100-130 nm for both biofilms. The mesh size of the biofilm matrix and the size of the channels in between the bacteria of the clusters are two factors which likely play a role in the exclusion of the larger particles. For B. multivorans, a sharp drop in the penetration into the clusters is seen for particles larger than 130 nm while for P. aeruginosa, a more gradual decrease in penetration could be observed. The overall penetration of the nanoparticles was slightly lower for P. aeruginosa than for B. multivorans. Based on these results, it could be concluded that nanocarriers of about 100 nm and smaller are good candidates to improve the treatment of chronic pulmonary biofilms in CF patients. Furthermore, the confocal microscopy method demonstrated here is a useful tool to assess the penetration of nanomedicines in biofilm clusters. Such information is important to optimize nanomedicine formulations for the treatment of biofilm infections.