Facile construction of antibiotics-loaded glucose-capped gold nanoparticles for in vitro antimicrobial and treatment and care of severe Pneumonia

From the perspective of gold nanoparticles (GNPs)′ potential antibacterial applications, we demonstrate the fabrication, characterization, and effective antimicrobial activity of gentamicin (GM) and kanamycin (KAN) dynamically loaded to glucose-capped gold nanoparticles (g-GNPs). Infra-red (FT-IR) spectroscopy analyzed the synthesized GNPs and g-GNPs with encapsulated antibiotic drugs. Various spectroscopical methods characterized g-GNPs and drug-loaded nanoparticles. A minimum inhibitory concentration (MIC) and active curves in the Klebsiella pneumonia strain were used to evaluate the antibacterial activity of aminoglycoside-loaded g-GNPs, and the results showed that the nanoparticles had an appropriate antimicrobial effect on the clinical strain of the bacteria. There were bacteriostatic effects and an inhibiting effect on the growth of bacteria at doses of 33 to 65 μg ml−1 for each GM@g-GNPs and KAN@g-GNPs. With zones of Inhibition (ZOI) of 27 and 29 mm, g-GNPs-loaded antimicrobial medications demonstrated more antibacterial activity in well diffusion experiments than free nanomaterials and antimicrobial drugs, with ZOI of 21.00 and 10.00 mm, respectively. GM and KAN-loaded g-GNPs were examined using crystal violet assay for their antibiofilm activity. Findings suggested that the concentration of nanoparticles and anti-biofilm activity were directly linked. The cell membrane integrity was assessed in g-GNPs loaded with GM and KAN, and the results showed that nucleic acids and proteins released into the environment were significant. The inhibitory effects of GM@g-GNPs and KAN@g-GNPs on bacterial efflux pump (EP) was assessed, and the result displayed that all strains were sensitive to moderate concentration of NPs and highly sensitive to concentrations of 0.6 and 0.9 μg ml−1 of ethidium bromide (EtBr) and 64 to 128 μg ml−1 of GM@g-GNPs and KAN@g-GNPs. The findings demonstrate that all strains were responsive to moderate nanoparticle concentrations. The results inhibited the efflux pump performance of the drug-loaded g-GNPs. Therefore, the unique design of these nanoparticles (GM@g-GNPs and KAN@g-GNPs) improved the antimicrobial properties, which has great potential for the treatment and care of severe pneumonia.


Introduction
A leading source of illness and disability, nosocomial infections may be found in nearly every country around the globe. Additionally, they were the sixth most significant cause of mortality in industrialized nations such as the United States. Acute and chronic hospital infections are caused by several Gram-negative and positive bacteria, including Pseudomonas, Escherichia, Proteus, Klebsiella, Enterococcus, Staphylococcus, and Streptococcus species [1][2][3]. Biofilms on implanted medical devices were implicated in 60%-70% of nosocomial infections [4]. Profound bacteria cells generate a protective layer of different polymeric substances known as biofilms,

Fabrication of glucose-capped GNPs loaded with antimicrobial drugs
It was possible to make the gold nanoparticles by reducing Au 3+ ions in aqueous glucose dispersions, and the efficient strategy was used according to the published. A 0.05 M solution of HAuCl 4 ·3 H 2 O was added to a 0.03 M glucose solution to fabricate a conventional formulation [41][42][43]. To confirm that the reduction reaction had failed, the solution was agitated for more than 30 min and showed no sign of colour change. Once the resolution colour had stopped changing, a constant stream of NaOH aqueous solution (0.05 M) was poured into the system. When NaOH was added to the solution, the dynamics of the reactions for the fabrication of Au nanoparticles were studied using a UV/vis spectrometer. After 30 min, a TOA pH meter and a Combination Electrode precisely recorded the pH values of the Au colloid solution.
When it comes to loading drugs onto the nanoparticles, two solutions were used: one solution of acetic acid (2 mg ml −1 , pH 5.5) and the other solution of 2.0 mg ml −1 of distilled water. To prepare the TPP solution, 400 mg of each drug (Gentamicin and Kanamycin) were dispersed in PBS and diluted (1:2) before being combined with the TPP solution and agitated for 15 min. Finally, 5 ml of g-GNPs solution and the TPP (2 ml) drugs were mixed and agitated at room temperature. 20 min were spent ultrasonically dissolving the reaction mixture, and then another 20 min were spent spinning the milky solution at 4°C and 12.000 rpm to separate the solids. Afterwards, the precipitate was dialyzed three times in double-distilled water for 12 min to eliminate contaminants from the supernatant. It was then dried at 65°C for 10 h to remove any remaining antibiotics from the GNPs loaded with GM and KAN.

Characterization of nanodrug formulations
Field emission scanning electron microscopy (FESEM) and energy-dispersive spectroscopy (EDS) were performed with a Sigma 500 scanning electron microscope (Carl, Germany). Transmission electron microscopic (TEM) images were obtained with a Tecnai G20 transmission electron microscope (FEI, USA). The Fourier transform infrared (FTIR) spectra of the samples were recorded using a Nicolet 6700 FTIR Spectrometer (Thermo Fisher, USA). The FTIR spectroscopy was applied to characterize the functional groups of catalysts in 400−4000 cm −1 using a Nicolet 6700 FTIR Spectrometer (Thermo Fisher, USA). The absorption spectrum was obtained using UV-3600 spectrophotometers (Shimadzu, Japan). Dynamic light scattering (DLS) and ZS90 Zetasizer Nano series Nano-ZS (Malvern Instruments Ltd, UK) instruments were used to calculate the nanosize and analyze the zeta potential.

Antimicrobial properties 2.4.1. Bacterial strains
The collection of samples was serially diluted, and the isolates were morphologically and microbiologically characterized as Klebsiella pneumoniae (K. pneumoniae). The isolated culture was identified as Klebsiella pneumoniae from MTCC and maintained by a subculture process to synthesize gold nanoparticles.

Examination of minimum inhibitory concentrations (MIC)
Microbicide susceptibility assessment was carried out using the CLSI technique to determine MIC and MBC of bacteria exposed to GM@g-GNPs and KAN@g-GNPs nanoparticles. We started by growing all the bacteria fresh in Mueller-Hinton broth (MHB), and later we used that culture to produce 0.5 McFarland standards for each strain in PBS. GM@g-GNPs and KAN@g-GNPs (10.250 μg ml −1 ) were prepared in dimethyl sulfoxide (DMSO) and then diluted with 2:5 in DD-water until they achieved a concentration of 4097 μg ml −1 (4-folds). Four-fold agents diluted in 100 μl of 96-well panel broth were cultured for 24 h at 37°C with 100 μl of the bacterial suspensions. Bacterial growth inhibition was measured using MIC. The minimal bacterial concentration (MBC) was defined as the concentration at which no observable growth of bacteria could be observed in Muller Hinton agar (MHA) [38,44,45].

Assessment of time kills curve evaluation
The dynamic bacterial curves were studied in the GM@g-GNPs and KAN@g-GNPs. Mueller-Hinton broth was initially used to cultivate the bacterial culture (MHB). The bacterial cells were extracted by centrifugation at 10.000 rpm for 10 min during the mid-exponential growth phase. Bacteria were rinsed thrice with DD-water, and standard 0.5 McFarland 2×10 5 CFU ml −1 was obtained after every strain was washed with the PBS. In 96well plates, diluted bacteria were combined with a variety of quantities of GM@g-GNPs and KAN@g-GNPs versus free drugs (1.024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μg ml −1 ), which were incubated at 37°C and counted after an h. No treatment was administered to the samples used as controls. OD 600 nm was determined during 18 h with an ELISA (ALLSHENG AMR-100 microplate reader). The examined bacteria strains were removed to MHA plates and cultured at 37°C for 24 h due to the antibacterial activity of various drugs [46]. The following formula was used to get the total number of bacterial colonies: Relative viability (%) = (Control count-treated samples count)/Control count × 100.

Investigation of agar well diffusion method
Agar well diffusion was employed to explore the antibacterial properties of GM@g-GNPs and KAN@g-GNPs. The bacteria were suspended in PBS to achieve a population of 2×10 3 CFU ml −1 according to the 0.5 McFarland standard [13,47,48]. Separate suspensions of bacteria were injected onto the nutrient agar's surface. A sterile cork borer was used to create wells on the agar surface with a diameter of 6 mm, and NPs solution at concentrations of 4,096 μg ml −1 was poured into each well in increments of 50, 100, and 150 μl each −1 well. Plates were then incubated for 24 h at 37°C to measure the wells' inhibition zone (mm) size. For the positive control, we utilized gentamicin (10 μg disc −1 ), kanamycin (30 μg disc −1 ), and PBS (15%).

Analysis of biofilm inhibition assay
At various concentrations (20, 40, 60, and 80 μg ml −1 ) of GM@g-GNPs and KAN@g-GNPs, a quantitative antibiofilm inhibitory assay was performed in a 96-well plate. Bacteria were grown in a new culture, and 0.5 MacFarland standard 2×10 4 CFU ml −1 were generated from every strain in tryptic soy broth (TSB). Each well contained 100 μl of bacterial cell culture versus a different NP concentration. The plates were kept at 37°C for 24 h. Positive (C+) and negative controls, free cell suspension, and NPs without bacterium induction were used in the study. The media was removed after incubation, and the wells were cleaned twice with sterilized distilled water to eliminate any single cells that may have formed. 1% crystal violet aqueous (w/v) solution was poured into each well and allowed to sit for 20 min before being removed [49][50][51]. It was determined that the reaction mixture had an absorbance more significant than 575 nm utilizing an ELISA (ALLSHENG AMR-100 microplate reader).

Assessment of the integrity of cell membranes
The bacterial cell membrane's integrity may be determined using the OD 260 and OD 280 nm wavelengths, which quantify the release of nucleic acid and protein from within the cell. Each flask was incubated with 65 μg ml −1 of GM@g-GNPs and KAN@g-GNPs, which were distributed to each flask and incubated with 250 rpm shaking. For the control sample, no nanoparticles were used in the incubation process. To remove 1 ml of the sample every 1 h, the flask was emptied and centrifuged at 5000 rpm for 4 min, and then the supernatants from the centrifuge were cleaned instantly.

Measurement of efflux inhibition
The inhibition activity of GM@g-GNPs and KAN@g-GNPs on the efflux pump was investigated using the approach we previously described. This study used three distinct EtBr concentrations: 0, 0, 5, and 8 μg ml −1 of trypticase soya agar (TSA). GM@g-GNPs and KAN@g-GNPs were given to each plate in 32, 64, and 128 μg ml −1 quantities. This was followed by 24 h of incubation at 37°C in PBS with an incubation standard solution (equal to 0.5 McFarland) applied on the TSA surface in a cartwheel pattern. After the plates had been incubated, the fluorescence of each bacterial strain was examined using an ultraviolet (UV) trans-illuminator [12, 52, 53].

Physicochemical characterization of nanoparticles
Although glucose is well known for reducing sugar, it does have limited reduction ability at ambient temperature. However, glucose can effectively reduce Au 3+ ions into Au 0 upon adding a small amount of NaOH aqueous solution; this was confirmed by visual observation and accurate UV/vis determination [54,55]. After the reduction process, as shown in figure 2(A), the solution turned red wine in colour, indicating the fabrication of Au nanoparticles. The figure 2(A) also shows the UV/vis absorption spectra showing reaction formation. HAuCl 4 absorption peak at 290 nm gradually decreases and disappears, as shown in figure 2(A), when NaOH aqueous solution is continuously added to the system. This disappearance co-occurs with the appearance of a new distinctive maximum absorption resulting from the surface-plasmon resonant frequency of nm size Au particles. The surface-plasmon absorption peak shifts from 537 nm to 525 nm with a rise in pH, which may be attributed to the formation of smaller Au nanoparticles. An absorption band at 3304 cm −1 in the FT-IR spectra ( figure 2(B)) confirms the existence of glucose as a critical component of g-GNPs. A high-frequency shift in the -OH stretching absorbance peak, found at 3304 cm −1 , suggests a close interaction between the Au nanoparticles and glucose. It has been determined that the bending vibration of -OH, as measured by FT-IR, is responsible for the unique signal at 3319 cm −1 indicated in g-GNPs ( figure 2(B)). Degraded hydrogen bonds of the chelate type can be displayed in the 3501-3201 cm −1 range. Deformation vibrations of the hydroxyl group may be exhibited in the 1395-1019 cm −1 range in g-GNPs.
The drugs capped gold nanoparticle spectra are displayed in figure 2(B). The detected peak at 3461 cm −1 is attributed to -OH vibrations, whereas the typical peak at 3301 and 3121 cm −1 is attributed to -NH vibrations. In this study, the C-H stretching vibrations of methyl and methylene may be found at 2961, 2871, and 1461 cm −1 . The prominent peaks at 1671 and 1621 cm −1 in gold correspond to -NH vibration, and the blue-shift to 1661 to 1611 cm −1 in gold. An amino group's C-N stretching vibration may be found at 1341 cm −1 , whereas CO stretching vibrations are demonstrated at 1201, 1113, and 1051 cm −1 . But the results show that g-GNPs may be successfully loaded with a drug ligand. The figure 3 depicts the TEM and SEM images of gold nanoparticles and antibiotics loaded gold nanoparticles' size and shape. As we anticipated from the absorption data and FT-IR evidence, the characteristic spherical GNPs are observed with a comparatively narrow particle size distribution. In addition, well-organized GNPs also appear in the image. Further, we effectively loaded gentamicin (GM) and kanamycin (KAN) into glucose-capped gold nanoparticles (g-GNPs), showing the morphological evidence in figure 3. Thus, GNPs, GM@g-GNPs, and KAN@g-GNPs sizes as measured by SEM and TEM are in good agreement and are 40-80 nm, suggesting that glucose capped on antibiotics reduced gold nanoparticles. Such size distribution analysis of glucose capped on antibiotics reduced gold nanoparticles confirms that particles are well dispersed ( figure 3). The surface modification can affect the surface charge of nanoparticles; this determines the dispersion and aggregation of colloidal solution at a given pH value. The GNPs used in this work were negatively charged, with an average ζ-potential value of −2492±5.3 mV.
According to the ED structure of these gold nanoparticles, the face-centred-cubic (fcc) organized gold and these Au nanoparticles are crystalline (JCPDS 04-0784). Furthermore, the x-ray diffraction (XRD) pattern of Au nanoparticles is presented in (figure 4(A), in which the diffraction peaks for the (002), (101), and (200) lattice planes appear clearly. Surface-enhanced Raman scattering/spectroscopy (SERS), a vibrational spectroscopic approach that offers information on molecule position, has attracted considerable attention for chemical and biological sensing and imaging. The high Raman signal at 1165 cm −1 may be due to C-H out of plane bending  since the peak at 1032 cm −1 results from C-H in-plane bending ( figure 4(B)). A C-O-C bond may cause the medium-sized Raman signal to be displayed at 968 cm −1 , while the orientation of C-O groups causes the peak at 1116 cm −1 . It has been reported that several bands at 1264 cm −1 have been ascribed to complicated CH 2 OH modes. While the optical characteristics of semiconductor and insulator nanomaterials are strongly influenced by their size and form, the optical metallic substrate nanoparticles are more shape-and size-dependent ( figure 4(B)). Gold nanoparticles (g-GNPs) fabricate may be more potent for therapeutic systems because of their customizable SPR capabilities and good SERS enhancement abilities. Because of their unique structure, these particles are superior candidates for optical imaging-based disease therapeutic applications.

Minimum inhibitory effect of nanodrugs based antibiotics
To measure the efficiency of antibiotics in g-GNPs, we used them in 96-well plates. The results showed that GNPs with caps on their mechanism of action were effective against bacteria. The results demonstrate that loaded antibiotic drugs to g-GNPs had a substantially more significant antibacterial impact than free GNPs and antimicrobial drugs against all examined strains [56][57][58]. Comparatively, free antibiotic (gentamicin) as control was 65 to 129 μg ml −1 , demonstrating that GM@g-GNPs had lower MIC. Similar results were shown in MBC, which displayed MHA plates less growth after plating 65 μg ml −1 of the GM@g-GNPs-treatment with bacterium strains (less than six colonies). MIC of free GNPs ranged from 32 to 256 μg ml −1 in examined strains, as the minimum inhibitory concentration (MIC) of KAN@g-GNPs was between 16 and 129 μg ml −1 . We established the effect of nanodrug-based antibiotics on bacterial growth by running an experiment with a dynamic growth curve using antibiotics with g-GNPs loaded at various concentrations. The results showed that this effect was substantial (figures 5(A)-(D)). GM@g-GNPs and KAN@g-GNPs at 33 μg ml −1 each demonstrated a bacteriostatic impact on a bacterial strain. There were no effects on bacteria when the nanoparticles were given at a 64 μg ml −1 concentration. According to our results, GM@g-GNPs and KAN@g-GNPs demonstrated considerable growth inhibition against the clinical strain of K. pneumoniae compared to the free antibiotics and GNPs used. A low concentration of nanodrugs formulation caused a delay in bacterial growth, and bacterial growth was entirely prevented when the concentration of nanoparticles was increased. In other words, no growth was detected after 24 h at a concentration of 1.024 μg ml −1 .

Well diffusion assessment
According to the well diffusion technique, the antibacterial activity of GM@g-GNPs and KAN@g-GNPs was shown to be superior to that of free antibiotic and gold nanoparticles, which were examined at 40, 80, and 120 μg ml −1 in the presence or absence of nanoparticles ( figure 6(A)). Increasing the concentrations by two-fold (80 μg ml −1 ) resulted in a 19-and 21 mm inhibition zone ( figure 6(B)). It was also shown that with a three-fold increase in nanodrug concentration, the area of Inhibition rises from 18 to 23 mm in the presence of clinical K. pneumoniae. At 40, 80, and 120 μg ml −1 concentrations, this value was also found for free GNPs, with 9-, 18-, and 21-mm inhibition zones, respectively. We employed gentamicin and kanamycin discs as positive controls, and the ZOI values on those discs were 9 mm for both samples. There was no suppression of bacterial growth in the negative control (15% PBS).

Anti-biofilm activity of nanodrugs formulations
The development of bacterial biofilms is critical for bacterial infection, and we have demonstrated here that nanodrugs have suitable antibiofilm action against bacterial strains KP2 and KP3. At 80 μg ml −1 concentrations of GM-g-GNPs, Klebsiella pneumoniae biofilm was destroyed (OD 0.04 ). Treatment with GM-g-GNPs at a 20 μg ml −1 concentration did not influence the formation of biofilms ( figure 7(A)). The figures 7(B)-(C) shows no biofilm formation was revealed at 80 μg ml −1 of KAN@g-GNPs in both strains. At 20 to 60 μg ml −1 concentrations, KAN@g-GNPs had only a minor impact. There was no growth in the negative control (nanodrugs) at OD0.02 for the positive control (bacteria without agents). GM@g-GNPs possess powerful antibiofilm properties, as demonstrated by these data. These positive-charged antimicrobial agents (GM and KAN) may be capable of binding to negative-charged materials in the biofilm pattern, causing an antibiotic penetration into the biofilm matrix. However, cationic gold can disrupt the negatively charged, hindering biofilms and bacterial surface production. GM@g-GNPs and KAN@g-GNPs are examples of integrated structures that might help antibiotics penetrate the biofilm matrix.

Cellular integrity in the presence of NPs
DNA and protein leakage from the cell membranes of GM@g-GNPs and KAN@g-GNPs-treated strains was assessed at OD 260 and OD 280 nm, respectively, to evaluate the cell membrane integrity strains studied (figure 8). Intracellular substances began to flow out of bacteria after 4 h of treatment with nanoparticles. Protein and nucleic acid adsorption in KAN@g-GNPs were examined in the OD 0.086 and 0.178 , and the control with OD: 0.03 and 0.076, respectively, after 2 h of incubations. Cellular content leakage was displayed at concentrations of 0.97 and 0.211 for protein content after 2 h of incubation in the occurrence of GM@g-GNPs, and then in the control samples with OD 0.0238 and OD 0.092 , respectively. As a result, it can be concluded that extending the incubation period led to more bacterial cell wall breakdown, which in turn led to more intracellular leakage. Antibiotics and g-GNPs may have a synergistic impact because of the electrostatic contact among polycationic constructions of GNPs and the bacteria's surface, LPS, which plays a critical role in antimicrobial activity. Additionally, because Gram-negative bacteria have a more negatively charged cell surface than Gram-positive bacteria, leaking intracellular content and proteinaceous components can occur after the cell membrane has been destroyed and destroyed. Antibacterial properties will be enhanced because of this strategy.
3.6. Inhibition effects of nanodrugs on efflux pumps g-GNPs were loaded with various doses of antibiotics and examined for their inhibitory impact on the efflux pumps (EPs) in the occurrence of EtBr, which functions as a substrate for the efflux pumps (EPs). g-GNPsloaded antibiotics may give access to or raise the local concentration of antibiotic drugs, as opposed to free drugs, based on the accumulation of nanodrugs in the bacterial membrane and cytoplasm. The drug efflux pumps are the primary mechanism through which bacteria like K. pneumoniae become resistant to antibiotics. AcrAB and kexD from the resistance/nodulation/cell division (RND) family are functional in K. pneumoniae, the kdeA efflux gene from the MATE family, and the kmrA gene from the major facilitator superfamily, and kpnEF from the small multidrug resistance (SMR) family. In addition, nano-drug-based antibiotics may damage cell membranes, resulting in the suppression of antibiotic action [59,60]. According to the findings, both antibiotics  incorporated into g-GNPs inhibited the efflux pumps of bacteria. A dose of 33 μg ml −1 KAN@g-GNPs and 0.2 μg ml −1 of EtBr reduced efflux pump efficiency in bacterial strain, which is interesting. Furthermore, the efflux pump in reference strains and wild-type were blocked by KAN@g-GNPs at 65 and 129 μg ml −1 and EtBr at 0.5-0.8 μg ml −1 . The inhibitory impact of GM@g-GNPs was also investigated because of its possible influence on the bacterial efflux technique, and the results suggested that GM@g-GNPs have much higher antimicrobial action on bacteria efflux pumps than KAN@g-GNPs formulations. At EtBr concentrations of 0.2-0.5 μg ml −1 and nanoparticle concentrations of 32-64 μg ml −1 , the GM@g-GNPs limit bacterial growth appropriately. At EtBr concentrations of 0.9 μg ml −1 and 129 μg ml −1 , the GM@g-GNPs completely suppress bacterial growth (figure 9). GNPs have been shown to impede DNA synthesis, followed by a reduction in mRNA synthesis because of their interactions with the outer cellular components, the cytoplasmic membrane, and the cytoplasmic constituents. GM@g-GNPs and KAN@g-GNPs, loaded with antibiotics, can destroy the bacterial cell wall. This is because there aren't any physical barriers between the two worlds. Antibiotics encased in g-GNPs backbones will have unrestricted access to their target areas. Drug encapsulation efficiency (EE), release profile diversity, and suitability for a wide range of drugs are all characteristics of gold nanoparticles.

Conclusion
Gold nanoparticles are now being evaluated for several biomedical applications because of their excellent biocompatibility, bioactivity, and degradability, essential in microbiology. In this work, aminoglycoside antimicrobial agents are incorporated into GNPs via a reduction process between GNPs scaffolds. There were antimicrobial effects on clinical samples of K. pneumoniae demonstrated by GM@g-GNPs and KAN@g-GNPs, both of which were loaded with gold nanoparticles. Consequently, the unique design of GM@g-GNPs and KAN@g-GNPs enhanced the antimicrobial properties, which has excellent potential for the treatment and care of severe Pneumonia.