Volume 30, Issue 3 (5-2023)
RJMS 2023, 30(3): 229-243 |
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Research code: IR.IAU.PS.REC.1400.137
Ethics code: IR.IAU.PS.REC.1400.137
Clinical trials code: IR.IAU. TMB.REC. 26.199
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Ashkezari S, Falsafi S, Mohebbi S, Mirzaie A, Moosavi Niri N. Formulation and preparation of naproxen loaded nanoniosomes: Evaluation of antimicrobial and anti-biofilm effects on clinical strains of Staphylococcus aureus and analysis of biofilm gene expression: An Experimental Study. RJMS 2023; 30 (3) :229-243
URL: http://rjms.iums.ac.ir/article-1-7315-en.html
URL: http://rjms.iums.ac.ir/article-1-7315-en.html
Department of Biology, Parand Branch, Islamic Azad University, Parand, Iran , Amir_mirzaie92@yahoo.com
Abstract: (1366 Views)
Background & Aims: Staphylococcus aureus is one of the human pathogens that causes a wide range of diseases such as endocarditis, blood, bone, skin and soft tissue infections (1). One of the resistant strains of this bacterium is methicillin-resistant strains of Staphylococcus aureus (MRSA), which has been reported as a serious risk by the Centers for Disease Control and Prevention. One of the pathogenic mechanisms and antibiotic resistance of this bacterium is the formation of biofilm, which causes this bacterium to bind to different surfaces (2). Biofilm-forming strains have become resistant to many antibiotics, so that biofilm-forming strains form extracellular matrices that are immune to the immune system and antibiotics. Various genes are involved in biofilm production, one of which is the icaB gene, which plays a key role in the production of poly N-acetyl glucose amine and biofilm production. The design of new antimicrobial drugs for the treatment of this bacterium is important, so finding a suitable treatment option for the treatment of infections caused by this bacterium is one of the challenges of researchers (3). Recent advances in nanotechnology have suggested alternative solutions, such as drug delivery systems, that increase drug specificity and efficiency (4). One of these drug delivery systems is nanosystems, which are composed of bilayer nonionic surfactants. Due to the importance of nanosystems, the aim of this study was to synthesize nanoparticles containing naproxen, to investigate their physicochemical properties and their antimicrobial and antifouling effects against Staphylococcus aureus strains.
Methods: Nanosomes containing naproxen were synthesized by thin layer hydration method. The confinement efficiency is indicative of the drug encapsulated in the nanosystem structure relative to the drug used. For this purpose, the nanonosomal formulation was first centrifuged at 4 ° C at 14000 g for 45 minutes. The nanosystem containing the drug precipitates and the free drug remains in the supernatant. The absorbance of the supernatant sample at 270 nm was read by a spectrophotometer and the amount of free drug was calculated from the initial value.
The Physico-chemical characteristics of prepared nanoniosome encapsulated naproxen was determined using scanning electron microscopy (SEM), Dynamic light scattering (DLS). The in vitro drug release study was done using dialysis bag (6). The Staphylococcus aureus strains were recovered from 100 clinical samples and their antibiotic resistance patterns were studied using disk diffusion method. The antibacterial activity of nanoniosome loaded naproxen and free naproxen were investigated using well diffusion and micro-dilution methods (7). The icaB biofilm gene expression analysis in S. aureus isolates which are treated with nanoniosome loaded naproxen and free naproxen were examined using Real Time PCR methods (8).
Draw diagrams and Statistical analysis was performed by GraphPad Prism software version 7 and SPSS version 23, and one-way analysis of variance was used for statistical analysis and p <0.05 was considered significant.
Results: In this study, using different molar ratios of surfactant, cholesterol and drug, different formulations of nanosystems containing naproxen were synthesized. The optimal niosome size synthesis was measured by DLS method. The results of electron microscopy (SEM) show that the synthesized nanonosomes have a spherical structure. In this study, the dialysis bag method was used to evaluate the drug release pattern. Figure 2 shows the pattern of naproxen release from nanosystems and free naproxen over 72 hours.
Out of 100 clinical specimens, 15 specimens of Staphylococcus aureus were isolated and identified using microbiological methods. The results of antibiotic resistance profile test showed that out of 15 strains, 10 strains were methicillin resistant (MRSA).
The antibacterial activity of nanoniosome encapsulated naproxen and free naproxen showed that the MIC was reduced by 2 to 4 times. The results of the well diffusion method showed that nanoniosomes containing naproxen had more significant antimicrobial power than free naproxen, so that the diameter of the growth inhibition zone increased with increasing nanosystem concentration. Real-Time PCR was used to evaluate the effect of nanoniosoms containing naproxen and free naproxen on icaB biofilm gene expression in Staphylococcus aureus strains. Also, after treating Staphylococcus aureus strains with naproxen-containing nanosystem sub-inhibitory concentration, it was shown that the expression of icaB gene was significantly reduced compared to the 16S rRNA reference gene (p <0.05).
Conclusion: In this study, naproxen was encapsulated as a drug compound in the nanoniosome structure and its physical and chemical properties including size, morphology, drug enclosure percentage and drug release were studied. The results of this study showed that the synthesized nanoniosome in the optimal formulation had a spherical shape, the average size was 125.3 nm with a confinement percentage of 66.84%. Drug release results also showed that naproxen in the formulated form in nanonosomes has a much slower release pattern than free naproxen, which is a suitable feature of a drug delivery system. The results of the antimicrobial test showed that naproxen-containing nanoniosome had more significant antimicrobial effects than free naproxen compared to free naproxen, reducing the MIC by 2 to 4 times. One of the antimicrobial mechanisms of nanoniosome containing naproxen is the fusion of nanoniosome with bacterial cell membranes, which can deliberately release the drug into the cytoplasm of the cell and cause bacterial cell death (10). The results of this study showed that nanoparticles containing naproxen have more significant anti-biofilm effects than free naproxen compared to free naproxen and can significantly reduce the expression of biofilm gene (11). One of the reasons for the anti-biofilm effects of naproxen-containing nanosystems is the greater penetration of naproxen-containing nanosystems into the biofilm structure, which can cause the death of bacteria, a decrease in the number of bacteria, and the conversion of biofilms into planktonic cells (12).
Methods: Nanosomes containing naproxen were synthesized by thin layer hydration method. The confinement efficiency is indicative of the drug encapsulated in the nanosystem structure relative to the drug used. For this purpose, the nanonosomal formulation was first centrifuged at 4 ° C at 14000 g for 45 minutes. The nanosystem containing the drug precipitates and the free drug remains in the supernatant. The absorbance of the supernatant sample at 270 nm was read by a spectrophotometer and the amount of free drug was calculated from the initial value.
The Physico-chemical characteristics of prepared nanoniosome encapsulated naproxen was determined using scanning electron microscopy (SEM), Dynamic light scattering (DLS). The in vitro drug release study was done using dialysis bag (6). The Staphylococcus aureus strains were recovered from 100 clinical samples and their antibiotic resistance patterns were studied using disk diffusion method. The antibacterial activity of nanoniosome loaded naproxen and free naproxen were investigated using well diffusion and micro-dilution methods (7). The icaB biofilm gene expression analysis in S. aureus isolates which are treated with nanoniosome loaded naproxen and free naproxen were examined using Real Time PCR methods (8).
Draw diagrams and Statistical analysis was performed by GraphPad Prism software version 7 and SPSS version 23, and one-way analysis of variance was used for statistical analysis and p <0.05 was considered significant.
Results: In this study, using different molar ratios of surfactant, cholesterol and drug, different formulations of nanosystems containing naproxen were synthesized. The optimal niosome size synthesis was measured by DLS method. The results of electron microscopy (SEM) show that the synthesized nanonosomes have a spherical structure. In this study, the dialysis bag method was used to evaluate the drug release pattern. Figure 2 shows the pattern of naproxen release from nanosystems and free naproxen over 72 hours.
Out of 100 clinical specimens, 15 specimens of Staphylococcus aureus were isolated and identified using microbiological methods. The results of antibiotic resistance profile test showed that out of 15 strains, 10 strains were methicillin resistant (MRSA).
The antibacterial activity of nanoniosome encapsulated naproxen and free naproxen showed that the MIC was reduced by 2 to 4 times. The results of the well diffusion method showed that nanoniosomes containing naproxen had more significant antimicrobial power than free naproxen, so that the diameter of the growth inhibition zone increased with increasing nanosystem concentration. Real-Time PCR was used to evaluate the effect of nanoniosoms containing naproxen and free naproxen on icaB biofilm gene expression in Staphylococcus aureus strains. Also, after treating Staphylococcus aureus strains with naproxen-containing nanosystem sub-inhibitory concentration, it was shown that the expression of icaB gene was significantly reduced compared to the 16S rRNA reference gene (p <0.05).
Conclusion: In this study, naproxen was encapsulated as a drug compound in the nanoniosome structure and its physical and chemical properties including size, morphology, drug enclosure percentage and drug release were studied. The results of this study showed that the synthesized nanoniosome in the optimal formulation had a spherical shape, the average size was 125.3 nm with a confinement percentage of 66.84%. Drug release results also showed that naproxen in the formulated form in nanonosomes has a much slower release pattern than free naproxen, which is a suitable feature of a drug delivery system. The results of the antimicrobial test showed that naproxen-containing nanoniosome had more significant antimicrobial effects than free naproxen compared to free naproxen, reducing the MIC by 2 to 4 times. One of the antimicrobial mechanisms of nanoniosome containing naproxen is the fusion of nanoniosome with bacterial cell membranes, which can deliberately release the drug into the cytoplasm of the cell and cause bacterial cell death (10). The results of this study showed that nanoparticles containing naproxen have more significant anti-biofilm effects than free naproxen compared to free naproxen and can significantly reduce the expression of biofilm gene (11). One of the reasons for the anti-biofilm effects of naproxen-containing nanosystems is the greater penetration of naproxen-containing nanosystems into the biofilm structure, which can cause the death of bacteria, a decrease in the number of bacteria, and the conversion of biofilms into planktonic cells (12).
Type of Study: Research |
Subject:
Microbiology
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