Encapsulation and Characterization of Meropenem in Niosome Nanoparticles and Inhibitory Effects of Antibiofilm and Antibacterial on Methicillin and Vancomycin Resistant Strains of Staphylococcus Aureus


 Background: We aim to assess the antibacterial and anti-biofilm properties of niosome-encapsulated meropenem. Methods: After isolating S. aureus isolates and determining their microbial sensitivity, their ability to form biofilms was examined using plate microtiter assay. Various formulations of niosome-encapsulated meropenem were prepared using the thin-film hydration method, Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Inhibitory Concentration (MIC) were determined, and biofilm genes expression was examined. Drug formulations’ toxicity effect on HDF cells were determined using MTT assay.Results: Out of the 162 separated Staphylococcus aureus, 106 were resistant to methicillin. 87 MRSA isolates were vancomycin-resistant, all of which could form biofilms. The F1 formulation of neoplastic meropenem with a size of 51.3 ± 5.84 and an encapsulation index of 84.86 ± 3.14 was detected, which prevented biofilm growth with a BDI index of 69% and reduced icaD, FnbA, Ebps biofilms’ expression with p ≤0.05 in addition to reducing MBIC and MIC by 4-6 times. Interestingly, F1 formulation of neoplastic meropenem indicated cell viability over 90% at all tested concentrations. Conclusions: Results of the present study indicate that niosome-encapsulated meropenem reduces the resistance of Staphylococcus aureus MRSA to antibiotics in addition to increasing its anti-biofilm and antibiotic activity, and could prove useful as a new strategy for drug delivery.


Introduction
Staphylococcus aureus MRSA is among the most important hospital pathogens and an important factor contributing to hospital-acquired infections. This bacteria results in dangerous and purulent infections in patients and causes death in hospitalized patients (1). Staphylococcus aureus could cause a variety of infections such as septicemia, bacteremia, pneumonia, and infections in the skin, bones, and soft tissue (2). This bacteria is known to be the leading prevalent pathogen of hospital-acquired infections and is resistant to a wide variety of antibiotics such as methicillin (2). Since the mortality rate due to MRSA strains is quite high, the use of effective antibiotics started in the 1960s (3). The most important antibiotics selected for the experimental treatment of infections from Staphylococcus aureus's (MRSA) methicillin-resistant strains are meropenem and vancomycin (4). Meropenem (with the brand name of Merrem) is a broad-spectrum antibiotic used to treat various types of bacterial infections including pneumonia, meningitis, sepsis, and anthrax (5). Meropenem is usually prescribed as a strategic drug to treat patients suffering from systemic infections (6). According to previous research, using meropenem together with other antibiotics increases their strength against MRSA strains (7). However, meropenem overuse over the last decade has resulted in an increased population of enterococci with the meropenem resistance gene. Hence, there is a high probability of plasmids containing resistance gene transferring to Staphylococcus aureus strains through conjugation (8). Staphylococcus aureus strains resistant to methicillin have been identified since 1996 and are known to be a major challenge for healthcare systems (9). Besides, another reason for Staphylococcus aureus strains' resistance to antibiotics is biofilm formation which has become a great challenge in the field of hospital-acquired infections. Biofilms are cohesive microbial communities enclosed in an extracellular polymer matrix and form on animate or inanimate surfaces (10). Biofilms could result in many complications if formed on oil reservoirs, air-conditioning systems, and medical equipment such as prostheses and catheters. Besides, biofilms increase bacteria's resistance to antibiotics (11). Several genes are involved in bacterial biofilm formation (12). The first stage of biofilm formation is adhesion to surfaces which is facilitated by the bacterium's icaD gene binding. icaD (N-acetyl glucosamine transferase) makes up a major part of the exopolysaccharide matrix of the biofilm (13). Biofilm strength and maturity are then facilitated due to the expression of FnbA, Ebps elastin, and Bap fibronectin-binding genes (14). FnbA contains multiple fibronectin (Fn) substituents and binding regions, each one capable of joining both immobilized and soluble forms of Fn. This enables S. aureus to attack endothelial cells in vivo and in vitro with no need for additional factors (15). The elastin-binding protein (EbpS) is a 25 kDa cell surface protein coded by the Ebps gene. The binding of S. aureus to the 30 kDa N-terminal region of PG. 3 elastin that is the main component of elastic fibers' extracellular matrix leads S. aureus to colonize in the tissue (16).
Bap is a protein associated with a cell wall that involves in the formation of biofilm (17). The spread of microbial resistance and the slow pace of producing new antibiotics over recent years have made researchers use nanotechnology to develop more and newer antibiotics. Low-cost production and causing no environmental issues are among the advantages that have turned nanoparticles into one of the suitable candidates for bacterial strain inhibition (18).
Antimicrobial agent encapsulation in nanocarrier systems is among the most promising and effective ways that improve antibacterial activity as well as reducing side effects (19,20). Niosomes have recently found a wide use to improve selective drug delivery and antimicrobial drugs' therapeutic efficacy. Niosomes are bilayer structures whose nonionic surfactants make them water-soluble and enables them to carry high drug doses (21). Niosomes have distinct features that could be used for the encapsulation of various drugs (22). The use of niosomes as antibacterial nanocarriers has recently attracted great attention from researchers (23). Hence, the present study has selected Meropenem as a strong antibiotic against Staphylococcus aureus MRSA strains to reduce antibiotic resistance and increase antimicrobial effects. In fact, the purpose of conducting this study was to develop niosome-encapsulated meropenem with improved antimicrobial activity against Staphylococcus aureus MRSA strains. Genomic DNA of the bacterium was obtained using the instructions of the Sina Gene company extraction kit (Cinna Pure DNA KIT, Alborz, Iran), and the purity obtained at the wavelength of 260nm was confirmed by a spectrophotometer. The M-PCR test was conducted to detect biofilm decoding genes, icaD, FnbA, Ebps, Bap, and the gene resistant to vancomycin VanB through polymerase chain reaction using oligonucleotide sequences of specific primers indicated in Table 1. Final reaction volume (20µL) was considered to contain 12 µL PCR master mix (PCR buffer, MgCl2, dNTP, 0.2 units of Taq polymerase), 0.5 µL reverse primer, 0.5µL forward primer, 1µL template cDNA, and 6µL distilled water (Amplicon, Denmark). The PCR program was performed in the form of an initial denaturation at 95°C for five minutes and 35 initial denaturation cycles for one minute at 94°C, one minute of connection at 58°C, one minute of extension at 72°C, and a final extension at 72°C for five minutes. The amplified products were investigated through 1% agarose gel electrophoresis to detect the desired genes.

Formulation and encapsulation of meropenem in noisome
According to instructions provided by previous studies (25,26). noisome-encapsulation of meropenem was conducted through thin-film hydration and 4 drug compounds were prepared as follows. The first compounds were prepared. Tween40 mixed with cholesterol with respective molar ratios of 3:3:4 and was dissolved in 3ml of chloroform and methanol mixed with respective ratios of 2:1(Table1). After adding glass beads to all four compounds, drug compound solvents were evaporated using a rotary evaporator (Heidolph, Germany) for one hour at 60°C and 120rpm rotation.
Afterward, dried thin films were hydrated for one hour using a solution of meropenem dissolved in 10ml of PBS at 60°C with a speed of 120rpm to obtain different niosome formulations. The resulting particles were sonicated using a probe sonicator in an ice bath using SONOPULS ultrasonic homogenizers (amplitude: 25%, 200 wt) for five minutes to reduce the size of niosomes containing meropenem, and samples were stored at 4°C for next experiments. The averages size, size distribution, and zeta potential of meropenem loaded in noisome were obtained using dynamic light scattering (DLS) and ZetaPlas palladium electrodes (Brookhaven Instruments Corp., USA). Various newlyprepared niosome formulations were diluted two times using distilled water with a ratio of 20:1 to prevent multiple scattering as a result of interactions between particles, and analysis was conducted at 25°C with a 90-degree light scattering angle. Average z diameter and niosome multiple scattering index were determined and their zeta potentials were measured. Niosome-encapsulated meropenem particles were coated with a gold layer to generate electrical conductivity and were examined using a field scanning electron microscope (FESEM) device model MIRA3 (TESCAN, Czech Republic) and an XRD device model X 'Pert Pro (Panalytical, Netherlands).

Entrapment efficiency (EE)
Encapsulation efficiency was obtained by determining the amount of non-capsulate meropenem (free meropenem) in the formed niosomes. Noisome-encapsulated meropenem particles were isolated over an hour at 4°C in a refrigerated centrifuge at 1400rpm. The meropenem content of the supernatant was examined through ELISA Reader Stat

Evaluation of noisome-encapsulated release and stability
Dialysis was employed to examine meropenem's release of the noisome. The dialysis tube was soaked in distilled water for 24 hours. 0.5ml (10mg) of meropenem-loaded noisome was put in the dialysis bag and 0.5ml meropenem antibiotic aqueous solution containing 10mg meropenem was also used as a control sample. Dialysis bags were immersed in conical flasks in 75ml distilled water and were shaken at 50rpm in a water bath at 37°C. 5ml was withdrawn from the receptor medium at intervals of one, two, four, six, 12, and 24 hours and meropenem were measured in terms of spectrophotometry at 281nm. Aliquots of samples were replaced with a new medium at 37°C and diffusion profile was determined using various kinetic models. This method was used to monitor the stability of PG. 7 The wells of the 96-well plate were filled with 100μl drug sample and 00μl of the bacteria cultured in Müller Hinton Broth. The plates were incubated for 48 hours at 37°C and were stained with violet crystal after being washed. The tests were replicated two times and the mean MBIC was determined to be OD630 <0.1.

b. Analysis of biofilm gene expression
The expressions of the biofilm genes of icaD, FnbA, Ebps, and Bap were examined through polymerase chain reaction (qRT-PCR) using the specific primers indicated in Table 1. RNA was extracted from resistant MRSA bacteria using RNX-Plus kit (Sina gene, Iran) after 24 hours of being exposed to sub-MIC concentrations of free meropenem and niosome-encapsulated meropenem, and cDNA was fabricated based on the RNA extracted from treated and untreated    (Table 3). As demonstrated, F1 is of a smaller and better size and is associated with surfactant Span60's hydrophile-lipophile balance. Span60 has a hydrophile-lipophile balance of 4.7 while the corresponding value in Span40 is 6.7. Therefore, nanoparticles formulated with Span60 are smaller. Besides, the EE content of the F1 formulation is higher than other formulations which might be due to the surfactant used. A longer saturated alkyl chain is directly associated with the permeability of drugs in niosomes so that longer saturated alkyl chains will result in greater permeability. Span60 has a longer alkyl chain compared to Span40, which is why formulations incorporating Span60 have higher indexes and EE content is at its peak in F1 formulation. The polydispersity indexes (PDI) smaller than 0.3 indicate a suitable distribution of small nanoparticles, indicating the F1 formulation to be the optimal one given that it has the smallest PDI. As figure 1 demonstrates, SEM results indicated a uniform spherical shape in the niosome-encapsulated meropenem of F1 formulation with an average size of 51.3 which indicates the suitable diameter (<100) of this drug formulation.      Out of the examined bacterial strains, the six isolates of SM RSA 1, S MRSA 2, S MRSA 3, S MRSA 4, S MRSA 5 and S MRSA 6 as well as the standard S. aureus ATCC 33592 isolate were selected as biofilm-generating strains, and the ability of niosomes carrying meropenem in inhibiting the growth of resistant Staphylococcus isolates through adhesion to biofilm over a short period of being exposed to drug formulations was compared to that of free meropenem. Compared to the minimum biofilm inhibitory concentration (MBIC) study, biofilm growth inhibition condition was harder since biofilm was first treated for only two hours with drug formulations or free meropenem; biofilms were then washed and incubated in an anti-biotic free medium for 24 hours. This study was conducted in concentrations ranging from 1.2-2MIC for respective isolates. The concentration of drug formulations compared to free meropenem MIC was calculated for physical composition and results were reported as Biofilm Growth Inhibition percentage (BIG%, Figure   5).   which results in increased drug encapsulation (38). Span60's long saturated alkyl chain resulted in more preamble niosomes as an influential factor, resulting in multiplied drug encapsulation (39). Besides, Span60 surfactant has a lipophilic balance of 4.7 which is lower than that of Span40 (6.7), resulting in the formation of smaller vesicles (40).  (41). This study's controlled drug release profile indicate a biphasic pattern. Given the niosome bilayer structure, the drug might reside either in the center of the niosome within the two layers or at the surface of the niosome during encapsulation (42). Therefore, the drugs on the surface start being released over the first hours of release which results in the blast phase (fast drug release). After seven hours, surface drugs will have released and encapsulated drugs residing at the center of the niosome and on the bilayer membrane start being released, resulting in controlled drug released and the stable phase. On the other hand, the cholesterol present in the system stops the gel to lipid phase transfer in niosome systems which prevents the drug from leaking out of the niosomes, resulting in controlled drug PG. 20

PG. 16
released over the long term and longer efficient drug release (43). Because of its higher strength as well as good surface density, formulation F1 forms better links with cholesterol, resulting in a reduced drug delivery process. Figure   3 indicates that this formulation has the longest time possible for controlled drug release (312 hours). Besides, negative zeta potentials are because of the electrostatic repulsion between the particles according to previous research, resulting in higher niosome stability (44). Studying the values of zeta potential and stability revealed that formulation F1 had the most stable niosome-encapsulated-meropenem with a zeta potential of -65.29 ± 2.68 over 56 days at 4°C and 25°C among the formulations studied. Additionally, drug stability was higher at 4°C compared to 25°C, which might be due to lower niosome bilayer mobility at 4°C (45). As a negative factor, the time of drug immobility has a direct relationship with nanoparticle size and an inverse relationship with EE index. Longer drug immobility time results in larger nanoparticle size due to particles' accumulation or fusion, and the pressure resulting from nanoparticle accumulation on niosome bilayer might result in the diffusion of the layers and reduction in their EE (46 (62,63). Hence, the present study examined the impact of niosome-encapsulated meropenem sub-MIC concentration on icaD, FnbA, Ebps, and Bap biofilm genes, revealing a considerable decline in the expression of these genes compared to free meropenem. Besides, the F1 formulation had the highest impact on reducing gene expression and was therefore identified to be the optimal formulation. Reducing the expression of the aforementioned genes could result in the inhibition of transcription through a direct impact and cause a reaction between niosome-encapsulated meropenem and transcription factors which will result in the inhibition or reduction of such genes' expression. These results are consistent with that of Lawson et al. (2012), revealing that the impact of Nano-encapsulation on increasing the inhibition of biofilms and reducing biofilm gene expressions (64).
The exopolysaccharide matrix of biofilm is the main source of defense and resistance against direct contact between Staphylococcus aureus and antibacterial agents (65). Results of this study revealed that the formulation of niosomeencapsulated meropenem reduces the resistance of Staphylococcus aureus MDR bacteria through biofilm growth inhibition and resulted in increased antibacterial properties for meropenem as well as being able to reduce biofilm gene expressions and prevent the growth of biofilm. Abdelazizi et al. suggested that niosome-encapsulation of norfloxacin improves its anti-biofilm properties and MDR bacteria biofilm formation (66). The increased microbial

PG. 22
resistance to various antibiotics had highlighted the need for finding new antibacterial compounds that are non-toxic for mammalian cells (67). The present study examined the toxicity of various niosome-encapsulated meropenem formulations for HDF cells through the standard method of MTT, indicating a higher cell viability rate in groups treated with niosome-encapsulated meropenem compared to free meropenem. Formulation F1 of niosomeencapsulated meropenem indicated the lowest cytotoxicity compared to other formulations with cell viability of over 90% over 24 hours. These formulations' cytotoxicity was due to the use of various Span: Tween surfactant ratios with high biodegradability, making these formulations suitable candidates for encapsulating meropenem as well as other drugs (68).

General conclusion
Niosome-encapsulated meropenem is a new approach for restoring meropenem characteristics with a low cost, giving meropenem distinct new properties such as increased targeted drug delivery, maintaining stability, and controlling drug release. According to this study, this drug formulation is more effective than free meropenem in treating infections due to more resistant Staphylococcus aureus isolates that are resistant to a variety of antibiotics, especially vancomycin and methicillin, and the present study is the first to report the impacts of encapsulating meropenem in niosome on biofilm formation and growth inhibition, biofilm eradication, and distinct antimicrobial activity against MRSA clinical isolates. As a general result, nisosmes are promising new drug systems increasing drugs' antibacterial effects whose antibacterial features depend on their formulation and composition. According to the present study's results, it could be inferred that niosome-encapsulation of meropenem increases its antibacterial and anti-biofilm activities against MDR and MRSA S. aureus strains and these formulations could be considered a new strategy for targeted drug delivery.

CONSENT FOR PUBLICATION
Not applicable.