Amikacin-loaded niosome nanoparticles improve amikacin activity against antibiotic-resistant Klebsiella pneumoniae strains

Abstract

Amikacin is an aminoglycoside antibiotic used in drug-resistant bacterial infections. The spread of bacterial infections has become a severe concern for the treatment system because of the simultaneous drug resistance bacteria and SARS-CoV-2 hospitalized patients. One of the most common bacteria in the development of drug resistance is Klebsiella strains, which is a severe threat due to the possibility of biofilm production. In this regard, recent nanotechnology studies have proposed using nanocarriers as a practical proposal to improve the performance of antibiotics and combat drug resistance. Among drug nanocarriers, niosomes are considered for their absorption mechanism, drug coverage, and biocompatibility. In this study, niosomal formulations were synthesized by the thin-layer method. After optimizing the synthesized niosomes, their properties were evaluated in terms of stability and drug release rate. The toxicity of the optimal formulation was then analyzed. The effect of free amikacin and amikacin encapsulated in niosome on biofilm inhibition were compared in multi-drug resistant isolated Klebsiella strains, and the mrkD gene expression was calculated. The MIC and MBC were measured for the free drug and amikacin loaded in the noisome. The particle size of synthesized amikacin-loaded niosomes ranged from 175.2 to 248.3 nm. The results showed that the amount of lipid and the molar ratio of tween 60 to span 60 has a positive effect on particle size, while the molar ratio of surfactant to cholesterol has a negative effect. The highest release rate in amikacin-loaded niosomes is visible in the first 8 h, and then a slower release occurs up to 72 h. The cytotoxicity induced by amikacin-loaded niosome is significantly less than the cytotoxicity of free amikacin in HFF cells (***p < 0.001, **p < 0.01). The mrkD mRNA expression level in the studied strains was significantly reduced after treatment with niosome-containing amikacin compared to free amikacin (***p < 0.001). It was confirmed that in the presence of the niosome, the amikacin antibacterial activity increased while the concentration of the drug used decreased, the formation of biofilm inhibited, and reduced antibiotics resistance in MDR Klebsiella strains.

Keywords: Amikacin; Antibiotic-resistance; Klebsiella pneumoniae; Niosome.

Fig. 1

Box–Behnken method for average diameter as a function of the parameters

Fig. 2

Box–Behnken method for PDI as a function of the parameters

Fig. 3

Box–Behnken method for encapsulation efficiency (EE) function of the parameters

Fig. 4

Morphological determination of optimized formulation. A SEM (Scanning electron microscopy), B dynamic light scattering (DLS)

Fig. 5

a Tween 60, b Span 60, c Cholesterol, d Niosome, e Amikacin, f) Niosome containing amikacin

Fig. 6

In vitro drug release profile of amikacin from Niosome at 37 °C

Fig. 7

Stability of optimum amikacin loaded niosomes stored during 60 days of storage at 4 ± 2 °C and 25 ± 2 °C, (*p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001)

Fig. 8

Biofilm formation values (OD 570) of MDR K. pneumoniae strains

Fig. 9

Anti-biofilm activity of optimum prepared niosome encapsulated amikacin using microtiter plate. The biofilm formation was quantified for each bacterial strain, and 200 µl of bacterial suspension was added to each well of 96 well plates, of which three wells were assigned to treating amikacin, three wells for treating amikacin- noisome, and three wells for positive control without treatment. Three wells contain only 200 µl broth media culture as negative control wells. (*p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001)

Fig. 10

PCR electrophoresis of entb, FimH, AcrAB, traT, arb, mrkD, irp-1, rmpA genes amplification

Fig. 11

mrkD gene relative expression in selected K. pneumoniae MDR strains is represented as fold difference between mrkD gene and 16S rRNA gene following treatment (*p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001)

Fig. 12

Cell viability at different concentrations of free amikacin and amikacin loaded niosome in HFF cells