Impact of the antibiotic-cargo from MSNs on Gram-positive and Gram-negative bacterial biofilms

Abstract

Mesoporous silica nanoparticles (MSNs) are promising drug nanocarriers for infection treatment. Many investigations have focused on evaluating the capacity of MSNs to encapsulate antibiotics and release them in a controlled fashion. However, little attention has been paid to determine the antibiotic doses released from these nanosystems that are effective against biofilm during the entire release time. Herein, we report a systematic and quantitative study of the direct effect of the antibiotic-cargo released from MSNs on Gram-positive and Gram-negative bacterial biofilms. Levofloxacin (LVX), gentamicin (GM) and rifampin (RIF) were separately loaded into pure-silica and amino-modified MSNs. This accounts for the versatility of these nanosystems since they were able to load and release different antibiotic molecules of diverse chemical nature. Biological activity curves of the released antibiotic were determined for both bacterial strains, which allowed to calculate the active doses that are effective against bacterial biofilms. Furthermore, in vitro biocompatibility assays on osteoblast-like cells were carried out at different periods of times. Albeit a slight decrease in cell viability was observed at the very initial stage, due to the initial burst antibiotic release, the biocompatibility of these nanosystems is evidenced since a recovery of cell viability was achieved after 72 h of assay. Biological activity curves for GM released from MSNs exhibited sustained patterns and antibiotic doses in the 2-6 μg/mL range up to 100 h, which were not enough to eradicate biofilm. In the case of LVX and RIF first-order kinetics featuring an initial burst effect followed by a sustained release above the MIC up to 96 h were observed. Such doses reduced by 99.9% bacterial biofilm and remained active up to 72 h with no emergence of bacterial resistance. This pioneering research opens up promising expectations in the design of personalized MSNs-based nanotherapies to treat chronic bone infection.

Keywords: Antibiotic-cargo; Biofilm; Biological activity curves; Mesoporous silica nanoparticles.

Figure 1

Structure and main chemical properties of the three antibiocis used in this work, namely, levofloxacin (LVX), gentamicin (GM) and rifampin (RIF).

Figure 2

TEM images of pristine MSNs before and after 9 days being soaked in PBS under physiological conditions.

Figure 3

Hydrodynamic diameter (D_H) by dynamic light scattering (DLS) for MSN and MSN-NH₂ after 48 h of incubation with DMEM supplemented with 10% FCS and PBS 1x (0% FCS). Zeta (ζ)-potential values for MSNs in different media are displayed in the inset table.

Figure 4

Amount of antibiotic loaded into MSN and MSN-NH₂ samples determined by elemental chemical analysis.

Figure 5

Released and active concentrations of antibiotic (LVX and GM) after release from MSN and MSN-NH₂ materials at different time periods determined by disc diffusion tests in planktonic E. coli bacteria cultures.

Figure 6

Released and active concentrations of antibiotic (LVX and RIF) after release from MSN and MSN-NH₂ materials at different time periods determined by disc diffusion tests in planktonic S. aureus bacteria cultures.

Figure 7

In vitro antibiofilm activity LVX and GM released from MSN and MSN-NH₂ materials at different times of assay in mature E. coli biofilms.

Figure 8

In vitro antibiofilm activity of LVX and RIF released from MSN and MSN- NH₂ materials at different times of assay in mature S. aureus biofilms.

Figure 9

Direct effect of the different nanosystems onto E. coli biofilm. Histograms represent the Log (CFU/mL) after incubation with the different nanosystems at two tested times (2 and 24 h). Control represents the bacterial control without any treatment. LVX represents the solely antibiotic incubated in the bacteria culture at maximum concentration release by the nanosystems. The effect of antibiotic-free nanosystems on E. coli biofilm is also displayed.

Figure 10

Cell viability studies of the samples at different concentrations for the MC3T3-E1 cell line with 1 day of exposure time. *, ** and *** vs. corresponding control without nanoparticles (ANOVA).

Figure 11

Cell viability studies of the samples at different concentrations for the MC3T3-E1 cell line with 4 days of exposure time. *, ** and *** vs. corresponding control without nanoparticles (ANOVA).