Multifunctional lipid-based nanocarriers with antibacterial and anti-inflammatory activities for treating MRSA bacteremia in mice

Abstract

Background: Bacteremia-induced sepsis is a leading cause of mortality in intensive care units. To control a bacterial infection, an immune response is required, but this response might contribute to organ failure. Kidneys are one of the main organs affected by bacteremia. Combination therapies with antibacterial and anti-inflammatory effects may be beneficial in treating bacteremia. This study aimed to develop nanostructured lipid carriers (NLCs) loaded with ciprofloxacin and rolipram that exert a combination of anti-methicillin-resistant Staphylococcus aureus (MRSA) and anti-inflammatory effects. Retinol was incorporated into the nanoparticles to transport retinol-binding protein 4 (RBP4) to the kidneys, which abundantly express RBP receptors. The NLCs were fabricated by high-shear homogenization and sonication, and neutrophils were used as a model to assess their anti-inflammatory effects. Mice were injected with MRSA to establish a model of bacteremia with organ injury.

Results: The mean nanoparticle size and zeta potential of the NLCs were 171 nm and - 39 mV, respectively. Ciprofloxacin (0.05%, w/v) and rolipram (0.02%) achieved encapsulation percentages of 88% and 96%, respectively, in the nanosystems. The minimum bactericidal concentration of free ciprofloxacin against MRSA increased from 1.95 to 15.63 µg/ml when combined with rolipram, indicating a possible drug-drug interaction that reduced the antibacterial effect. Nanoparticle inclusion promoted the anti-MRSA activity of ciprofloxacin according to time-kill curves. The NLCs were found to be largely internalized into neutrophils and exhibited superior superoxide anion inhibition than free drugs. Retinol incorporation into the nanocarriers facilitated their efficient targeting to the kidneys. The NLCs significantly mitigated MRSA burden and elastase distribution in the organs of MRSA-infected animals, and the greatest inhibition was observed in the kidneys. Bacterial clearance and neutrophil infiltration suppression attenuated the bacteremia-induced cytokine overexpression, leading to an improvement in the survival rate from 22% to 67%.

Conclusions: The dual role of our NLCs endowed them with greater efficacy in treating MRSA bacteremia than that of free drugs.

Keywords: Bacteremia; Ciprofloxacin; Methicillin‐resistant Staphylococcus aureus; Nanostructured lipid carriers; Rolipram; Sepsis.

Fig. 1

Determination of the antibacterial activity of free drugs and NLCs against planktonic drug-resistant MRSA:  a the time-killing curves of ciprofloxacin (0.5 µg/ml) and rolipram (0.2 µg/ml); and b the time-killing curves of ciprofloxacin (1 µg/ml) and rolipram (0.4 µg/ml). All data are expressed as the mean ± SEM (n = 3). MRSA, methicillin-resistant Staphylococcus aureus; OD, optical density; SEM, standard error of mean

Fig. 2

Effects of free drugs and NLCs on primary human neutrophils (6 × 10⁵ cells/ml): a neutrophil cytotoxicity assay by LDH determination; b the measurement of extracellular superoxide production; c the uptake of rhodamine 800-loaded NLCs by human neutrophils observed via confocal microscopy; and d the fluorescence intensity of rhodamine 800 in the neutrophils analyzed by flow cytometry. All data are expressed as the mean ± SEM (n = 3). * p < 0.05 as compared to control group. LDH, lactate dehydrogenase; DAPI, 4’,6-diamidino-2-phenylindole

Fig. 3

Ex vivo bioimaging of organs in the rats receiving intravenous iFlour 790-loaded NLCs: a the blank group (non-treated control); b Free iFlour 790; c iFlour 790-loaded NLCs containing 0.25% retinol; d iFlour 790-loaded NLCs containing 0% retinol; e iFlour 790-loaded NLCs containing 0.1% retinol; and f the NIR intensity of iFlour 790 in the organs. All data are expressed as the mean ± SEM (n = 6). GI, gastrointestinal tract; NIR, near-infrared

Fig. 4

In vivo effect of free drugs and NLCs on MRSA-infected mice during 4 days: a the survival rate of the mice determined by Kaplan-Meier curves; b MRSA CFU in organs; and c elastase distribution in organs observed by IVIS. All data are expressed as the mean ± SEM (n = 14−27 for survival rate and n = 5 for MRSA CFU and elastase analysis). CFU, colony-forming unit; MRSA, methicillin-resistant Staphylococcus aureus

Fig. 5

In vivo effect of free drugs and NLCs on MRSA-infected mice during 40 h: a MRSA CFU in organs; b elastase distribution in organs observed by IVIS; c IFN-γ expression in organs; d IL-1β expression in organs; e IL-6 expression in organs; f IL-17A expression in organs; g TNF-α expression in organs. All data are expressed as the mean ± SEM (n = 6 for MRSA CFU and elastase analysis). * p < 0.05 as compared to MRSA-infected group. CFU, colony-forming unit; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-17A, interleukin-17A; MRSA, methicillin-resistant Staphylococcus aureus; TNF-α, tumor necrosis factor-α

Fig. 6

The H&E staining of the organs in the mice with or without MRSA infection. MRSA, methicillin-resistant Staphylococcus aureus

Fig. 7

The histology of the organs in the mice with or without MRSA infection: a MRSA distribution determined by S. aureus Rosenbach antibody; and b neutrophil distribution determined by recombinant anti-Ly6G antibody. MRSA, methicillin-resistant Staphylococcus aureus