Loading of vancomycin and amikacin into the UIO-66-NH2@PEG

A solution for synthesizing UIO-66-NH₂ was prepared by dissolving 0.41 g of 2-amino terephthalic acid (purchased from Sigma-Aldrich, Germany) and 0.54 g of ZrCl4 in 31 mL of DMF at 25 °C. The mixture was consistently stirred until a transparent solution was obtained. The solution was then transferred to a Teflon-coated hydrothermal autoclave and heated at 120 °C for 24 h. It was then cooled back down to the surrounding temperature. The resultant particles were centrifuged three times and subjected to ultrasonic treatment in DMF and chloroform for 15 min to replace DMF and remove any unreacted materials. Five days after the initial preparation, the UIO-66 sample underwent a solvent exchange process. This involved daily sonication of the sample for 15 min, fully submerged in 15 mL of fresh chloroform, for five consecutive days. The nanoparticles were then pressure dried at 120 °C to eliminate any remaining solvents.

To integrate VAN and AMK (both from EXIR Company, Iran) into the UIO-66 -NH2, VAN and AMK, were initially dissolved in DMSO, and then mixed with PBS (Gibco, USA) to a concentration of 0.25 mg/ml. This solution was combined with 10 mg of the synthesized MOFs, and the resulting mixture was stirred for 24 h. Afterward, the mixture was washed three times with ethanol and distilled water, followed by centrifugation at 12,000 rpm for 20 min [27].

The VAN/AMK-UIO-66-NH2 particles were dispersed evenly in an aqueous solution of PEG (20 mg/mL) using ultrasonication (50 kHz, 100 W L-1) for duration of 5 min in a glass tube with a phenolic cap. Afterwards, the tube was wrapped in foil to protect it from light and shaken for 24 h. The VAN/AMK-UIO-66-NH2@PEG particles were separated by centrifugation at 4,000 rpm for 10 min. Following this, the particles were washed three times with deionized water. Finally, the particles were resuspended in deionized water and subjected to freeze-drying for 48 h.

Characterization of VAN/AMK-UIO-66-NH2@PEG

The morphology of VAN/AMK-UIO-66 -NH2@PEG was examined by SEM using a TESCAN VEGA 3SB instrument and TEM with a Philips CM30 instrument from the Netherlands. The hydrodynamic diameter and size distribution of the VAN/AMK-UIO-66-NH2 and VAN/AMK-UIO-66-NH2@PEG compound were measured using DLS with a Malvern Zetasizer Nano instrument (Malvern Instruments, Worcestershire).

Determination of the entrapment efficiency percent

The entrapment efficiency was assessed by calculating the concentration of VAN and AMK that were not trapped. The complementary medication was extracted from the UIO-66-NH2 and modified UIO-66-NH2 solution using Ultracel-30 K Millipore filters with a molecular weight cut-off (MWCO) of 30,000 Da. The formulation was placed in the inner compartment of the cell, with a volume of 500 µL. The setup was then centrifuged for 20 min at 4000×g at 4 °C, using a cooling centrifuge (Eppendorf® 580R centrifuge, Germany). The concentration of free drug in the exterior compartment of the equipment was determined using UV-visible spectrophotometry at 250 and 520 nm (JASCO, V-530, Japan). The EEpercent was calculated using Eq. 1 (Eq. 1) [27].

Equation 1: EE (%) = \(\:\frac\:\times\:100\)

In vitro release study of the formulations

The release of VAN and AMK from drugs-loaded UIO-66-NH2 and UIO-66-NH2-PEG was investigated using the dialysis diffusion bag method. Accordingly, 2 mL of the MOF suspension was placed into a dialysis bag and sealed.

The dialysis diffusion bag technique was used to evaluate the release of VAN and AMK drug-loaded UIO-66-NH2 and UIO-66-NH2@PEG. The molecular weight of the dialysis bag used was 12 kDa (Merck, Germany). Firstly, 2 ml of the MOF suspension was introduced into a dialysis bag and then sealed. The bag was immersed in 50 mL of PBS-SDS (pH 5.4 and 7.4, 37 °C), which served as the medium for releasing or receiving the substance. The bag was then placed on a magnetic stirrer set to 300 RPM. Samples were taken from the buffer compartment at predetermined intervals and replaced with fresh PBS-SDS (Merck, Germany). The OD of each sample was measured at 250 nm and 520 nm. The released VAN and AMK amounts were estimated using the equation derived from a standard curve. A graph was generated to illustrate the cumulative amount released over time [28].

Determination of the formulations physical stability

The stability of VAN/AMK-UIO-66-NH2 and VAN/AMK-UIO-66-NH2@PEG was evaluated for 60 days at two different temperatures (25 ± 2 °C and 4 ± 2 °C). At specific time intervals (14, 30 and 60 days), the structures’ dynamic particle size, PDI and EE% were measured and compared to fresh formulations.

Bacterial isolation and characterization

S. aureus isolates were isolated from 500 clinical samples based on their culture characteristics and biochemical tests. The bacteria were isolated from blood, sputum, and urine samples of patients hospitalized at Baqiyatallah Hospital in Tehran between April and September 2023. The biofilm formation ability of the S. aureus isolates was evaluated using Congo-red agar and crystal violet assays [28]. Additionally, the presence of the vanA, mecA, icaA, and icaD genes in the isolates was assessed using the PCR technique. The antimicrobial sensitivity of the isolates was determined by the Kirby-Bauer disk diffusion susceptibility test. The bacterial isolates that exhibited high biofilm formation potential, show resistance to vancomycin and carried the vanA gene (as VRSA isolates) were selected for further investigation.

Antibacterial activity of formulations

TheMIC of VAN, AMK, VAN/AMK, VAN/AMK-UIO-66-NH2, and VAN/AMK-UIO-66-NH2@PEG was measured using a conventional broth microdilution method with values ranging from 2 to 512 µg/ml. The samples were diluted in a 96-well microplate using Mueller-Hinton broth (MHB). Finally, bacterial suspensions were prepared with a turbidity of 0.5 McFarland by diluting in MHB and added to each well to yield a final concentration of 4–5 × 105 CFU/ml in wells. The samples were then incubated overnight at 37 °C. The MIC was defined as the lowest concentration at which no bacterial growth was observed. To establish MBC values, 10 µl samples from wells without visible growth were plated on MHA. After overnight incubation, colony counts were used to assess bacterial viability. The MBC was defined as the lowest compound concentration that resulted in the death of 99.9% of the bacterial inoculum [28].

Time-kill assay

The 96-well plate method was applied to conduct time-kill assay. The compounds VAN/AMK, VAN/AMK-UIO-66-NH2, and VAN/AMK-UIO-66-NH2@PEG were diluted to concentrations corresponding to their MIC values, following standard procedure. After adding 100 µl of each sample to a microtiter plate well that had previously been filled with 100 µl of a bacterial solution containing 105 CFU/ml, the plates were incubated at 37 °C. The OD at 600 nm was measured at 8, 16, 24, 32, 40, 48, 56, 64, and 72 h using a microplate reader (EPOCH, Japan). A bacterial growth curve was used as a positive control [29].

Biofilm formation inhibition assay

The ability of the VRSA isolate treated with VAN/AMK, VAN/AMK-UIO-66-NH2, and VAN/AMK-UIO-66-NH2@PEG to form biofilm was assessed using a 96-well microtiter plate technique, as previously described [30]. In brief, the isolates were cultured in 96-well microtiter plates for 24 h at 37 °C. The strains were then treated with MIC values of each compound for an additional 24 h at 37 °C. After overnight incubation, the plates were rinsed with PBS to remove non-adherent bacteria. Ultimately, the biofilms were treated with a 1%W/V CV solution and the stain was dissolved in 95% ethanol. The OD of each well was measured at 570 nm using an ELISA reader (Stat Fax 2100, USA). The average absorbance values of each sample were computed and compared to the average values of controls (untreated isolates).

The MBEC was used to assess the ability of the test compounds to disrupt pre-existing biofilms. The strains were allowed to form biofilms as shown in the biofilm formation assessment. Subsequently, the wells were rinsed with sterile PBS, and 200 µL of each tested concentration of VAN/AMK, VAN/AMK-UIO-66-NH2, and VAN/AMK-UIO-66-NH2@PEG were added. The plates were incubated for 24 h under aerobic conditions at 37 °C. The OD of the biofilms was then measured at 570 nm. The negative control consisted of uninfected Tryptic Soy broth (Oxoid, USA), whereas the positive control was a simple bacterial culture. The MBEC was defined as the concentration at which the average biofilm OD was equal to or lower than that of negative control. This experiment was conducted in duplicate [31].

Bacterial gene expression in treatment with the formulations

The study aimed to assess the mRNA expression levels of vanA, mecA, icaA, and icaD genes in VRSA isolates after being exposed to sub-MIC concentrations of VAN/AMK, VAN/AMK-UIO-66-NH2, and UIO-66-NH2@PEG formulations. This was done using qRT-PCR. Firstly, the target genes were identified in VRSA isolates using PCR. The primers used for qRT-PCR are provided in Table 1.

Table 1 Primers used in the current survey

First, total bacterial RNA was extracted following the protocol provided by the RNX-Plus extraction kit (CinnaGen, Iran). Complementary DNA (cDNA) was then synthesized using the RevertAid™ First Strand cDNA Synthesis Kit, (Fermentas, USA). Afterwards, NanoDrop was used to measure the concentration of the extracted cDNA. The gene expression levels of the examined genes were evaluated via q-RT-PCR using a Master Mix containing SYBER Green (Ampliqon, Denmark). The reactions was conducted in a 25 µl solution containing 1 µl of diluted cDNA, one µl of forward and reverse primers, and 12.5 µl of SYBR Green Master Mix.

The following steps were used to conduct the qRT-PCR assay: 95 °C for 60 s, 30 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 1 min, extension at 72 °C for 45 s, and a final extension at 72 °C for 10 min. The 16 S rRNA gene was employed as a reference control [32]. The 2 − ΔΔCt formula was applied to calculate the fold change in the expression of the target genes, normalized to the reference gene (16 S rRNA) and compared to the expression in the untreated sample.

Antioxidant activity

The DPPH test is used to evaluate the antioxidant characteristics of various substances. DPPH is dissolved in ethanol and transformed into its radical form. This radical form exhibits the maximum absorbance level at a wavelength of 517 nm. This radical reacts with antioxidant compounds, leading to its neutralization. As a result, the absorbance of the solution at 517 nm decreases, causing a color changes from purple to yellow.

This study explores the antioxidant activity of the target compound. To prepare the DPPH radical solution, 1 mg of DPPH was dissolved in 9.16 ml of ethanol. Various concentrations (12.5, 25, 50, 100, 200, and 400 µg/ml) of VAN/AMK, VAN/AMK-UIO-66-NH2 and VAN/AMK-UIO-66-NH2@PEG were prepared and mixed with an equal amount of the DPPH solution. The mixture was vortexed for 10 s and kept in dark environment at room temperature for 30 min. Subsequently, the absorbance of the sample was measured at 517 nm. Butyl hydroxy anisole (BHA) from Merck Germany was utilized as a positive control, while distilled water served as the negative control to compare the effectiveness of the formulations [33].

Statistical analysis

Statistical analysis was conducted using GraphPad Prism (GraphPad Software, San Diego, CA) and SPSS V.20. All tests were performed three times, and the findings were reported as the average values and corresponding standard deviations. The statistical variance analysis technique (ANOVA) was used to assess the differences between the groups. The confidence level was established at 95%, and the results of p < 0.05 were considered to have statistical significance.