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. 2016 Aug 1;10(4):190–194. doi: 10.1049/iet-nbt.2015.0034

Gentamicin‐gold nanoparticles conjugate: a contrast agent for X‐ray imaging of infectious foci due to Staphylococcus aureus

Azam Ahangari 1, Mojtaba Salouti 2,✉, Faranak Saghatchi 3
PMCID: PMC8676068  PMID: 27463788

Abstract

There is no optimal imaging method for the detection of unknown infectious foci in some diseases. This study introduces a novel method in X‐ray imaging of infection foci due to Staphylococcus aureus by developing a contrast agent based on gold nanoparticles (GNPs). GNPs in spherical shape were synthesised by the reduction of tetrachloroauric acid with sodium citrate. Then gentamicin was bound directly to citrate functionalised GNPs and the complex was stabilised by polyethylene glycol. The interaction of gentamicin with GNPs was confirmed by ultraviolet–visible and Fourier transform infrared spectroscopies. The stability of complex was studied in human blood up to 6 h. The stability of conjugate was found to be high in human blood with no aggregation. The biodistribution study showed localisation of gentamicin–GNPs conjugate at the site of Staphylococcal infection. The infection site was properly visualised in X‐ray images in mouse model using the gentamicin–GNPs conjugate as a contrast agent. The results demonstrated that one may consider the potential of new nanodrug as a contrast agent for X‐ray imaging of infection foci in human beings which needs more investigations.

Inspec keywords: drugs, nanomedicine, nanoparticles, nanofabrication, diagnostic radiography, microorganisms, diseases, polymers, ultraviolet spectra, visible spectra, Fourier transform infrared spectra, gold

Other keywords: gentamicin‐gold nanoparticle conjugate, contrast agent, X‐ray imaging, Staphylococcus aureus, disease, tetrachloroauric acid reduction, sodium citrate, polyethylene glycol, ultraviolet‐visible spectroscopy, Fourier transform infrared spectroscopy, human blood, Staphylococcal infection, X‐ray images, murine model, nanodrug

1 Introduction

Diagnosis of infectious foci and initiation of treatment at early stage of disease is critical for a favourable outcome. The detection of deep seated infections in some diseases such as intra‐abdominal abscesses, endocarditic and osteomyelitis is difficult and leads to delayed diagnosis and treatment [1, 2]. The most reliable method for early detection of infection is analysis of microbiological samples that is usually invasive and may be difficult or even impossible to perform in many cases [3]. On the other hand, imaging techniques such as ultrasound, computerised tomography scan and magnetic resonance imaging used in diagnosis of many diseases are of limited use due to insignificant anatomical changes in early stages of infection process and not specific for infection diagnosis [3, 4].

To date, nanoparticles (NPs) of various types have shown great promise as nanodiagnostic agents for infectious diseases [5]. NP technology based on fluorescent NPs (e.g. dye‐loaded NPs and quantum dots), magnetic NPs and metallic NPs (e.g. gold and silver NPs) have been successfully used to track and detect various infectious microorganisms [6].

However, there is no report about X‐ray imaging of infectious foci based on NPs conjugated with antibiotics as contrast agents. Within the last decade, there has been substantial interest in gold NPs (GNPs)‐based contrast agents in X‐ray imaging [7]. First, Hainfeld et al. [8] used GNPs as a contrast agent successfully in X‐ray imaging of EMT‐6 mammary tumour in mouse model in 2005. He announced the emergence of a new contrast agent with unique physical and pharmacokinetic properties over current agents to the world. Gold with a higher atomic number (Au: 79 versus I: 53) and a higher absorption coefficient (at 100 keV, gold: 5.16 cm2 g−1; iodine: 1.94 cm2 g−1) provides about 2.7 times greater contrast per unit weight than iodine [9, 10, 11].

In fact, GNPs have shown to match or exceed the performance of conventional iodinated contrast agents as expected from higher k‐edge energy of gold [12]. GNPs are an obvious choice due to their amenability of synthesis and functionalisation, less toxicity and ease of detection [13]. These properties make GNPs a very desirable metal as an ideal contrast agent for X‐ray imaging. On the other hand, the use of hybrid systems comprising of imaging agents and antibiotics present a promising approach for precise diagnosis and detection of infectious lesions [4]. The labelling of antibiotics with radionuclides was introduced about a decade ago by Solanki et al. in their research for developing the radiopharmaceuticals for the detection of infection sites in nuclear medicine. Theoretically, radiolabelled antibiotics would be incorporated and metabolised by the bacteria present in the infectious and, assuming that the uptake is proportional to the number of microorganisms, the radioactivity would accurately and specifically localise at the infection site [14]. Infected tissues exhibit extravasation of macromolecules, including plasma proteins and liposomes like tumour tissues. Clearance of macromolecules from tumour is so impaired that they remain in the tumour for a long time. This phenomenon has been characterised and termed as enhanced permeability and retention (EPR) effect [15]. The EPR effect, whilst recognised as a major breakthrough in anti‐tumoural targeting, has not yet been fully exploited in infection diagnosis. Shared pathophysiological pathways in both infection and cancer are evident and a number of novel nanomedicines have shown promise in selective and passive targeting of infection sites [16]. The aim of this study was to develop a new contrast agent for X‐ray imaging of infection foci due to Staphylococcus aureus based on GNPs conjugated with gentamicin.

2 Materials and methods

2.1 Materials

HAuCl4 ·3H2 O, trisodium citrate and gentamicin sulphate were purchased from Sigma, Aldrich, USA. Polyethylene glycol (PEG) (4000) and 4‐(2‐hydroxyethyl)‐1‐piperazine‐ethanesulphonic acid (HEPES) buffer were purchased from Merck, Germany. BALB/c mice (7–8 weeks old and 25–35 g weight) were purchased from Razi Vaccine and Serum Research Institute, Karaj, Iran. S. aureus ATCC 6538 was obtained as a lyophilised culture from microbial bank of Institute Pasteur, Tehran, Iran.

2.2 Synthesis of GNPs

GNPs were prepared by the reduction of tetrachloroauric acid with sodium citrate. Basically 50 ml of 0.01% HAuCl4 ·3H2 O solution is heated to boiling while stirring in a 100 ml beaker. Then a 450 μl of 1% of trisodium citrate solution is quickly added to the auric solution. The previously yellow solution of gold chloride turned wine red in colour. The size and morphology of GNPs were analysed by transmission electron microscopy (TEM). For TEM studies, the sample was prepared by placing a drop of colloidal solution of nanogold on a carbon‐coated copper grid and setting a completely dried drop by vacuum desiccators. The image of the sample was obtained using a TEM (JEOL model 1200 EX, University of Tehran). Then, surface plasmon resonance of GNPs was characterised using an ultraviolet–visible (UV–vis) spectrophotometer (Cary 100, Varian, CA) at the resolution of 1 nm from 400 to 900 nm [17, 18]. The concentration of GNPs was measured by an atomic absorption spectrometer (model AA240FS, VARIAN, Canada) [19].

2.3 Conjugation of GNPs with gentamicin

Conjugation of gentamicin with GNPs was carried out according to the method described by El‐Sayed et al. [18] with some modifications. Basically, the GNPs were diluted in 20 mm HEPES buffer at pH 5 to the final concentration with optical density of 0.8 at 520 nm. Forty microlitres of gentamicin (6 mg/ml) was added to 960 µl of HEPES buffer to form 1 ml of dilute solution. Then, 10 ml of the prepared gold solution was added dropwise to the dilute gentamicin solution while being stirred. After stirring for 5 min, the solution was left to react for 20 min. To prevent aggregation and increase stability properties in vivo, 0.5 ml of 1% PEG 4000 was added to gentamicin–GNPs conjugate and the mixture was stirred for 1 h. The final solution was centrifuged at 6000 rpm for 30 min and PEGylated gentamicin–GNPs pellet was redispersed in phosphate buffer saline (PBS) at pH 7.4 and stored at 4°C.

2.4 Gentamicin–GNPs characterisation

2.4.1 UV–vis spectroscopy

UV–vis spectroscopy (CARY‐100 BIO, Varian, USA) was used preliminary for investigating the interaction of gentamicin with GNPs. The spectrum ranged from 200 to 800 nm at the resolution of 1 nm [19, 20].

2.4.2 Fourier transform infrared (FT‐IR) spectroscopy

FT‐IR analysis was performed by Perkin‐Elmer FT‐IR spectroscopy (model FT/IR‐SDC300) for confirming the binding of NH2 groups of gentamicin molecules to GNPs. The spectrum ranged from 400 to 4000 cm−1 at the resolution of 4 cm−1 by making a KBr pellet with gentamicin and gentamicin–GNPs conjugate [17, 21].

2.5 In vitro stability in human blood

The samples of human blood were prepared and heparinised at the final concentration of 10 units/ml [12, 16]. 100 μl of gentamicin–GNPs conjugate was added to each millilitre of blood samples. The samples were mixed vigorously and incubated at room temperature for 6 h. Then, the samples were transferred to 1.5 ml eppendorf tubes and centrifuged at 2000 rpm for 15 min to precipitate red blood cells (RBCs). Next, the supernatant solution, which contained GNPs but no RBCs, was collected and transferred to a 1 cm cuvette [12]. The light absorption of samples was determined using a UV–vis spectrophotometer.

2.6 Biodistribution study

S. aureus was cultured in aerobic condition in nutrient agar medium for 16 h at 37°C. Then, a turbid suspension of viable bacteria containing 1.5 × 108 CFU/ml in 0.2 ml of normal saline was injected intramuscularly to the left lateral thigh muscles of four groups of five BALB/c mice. The right thigh muscle of each mouse was injected with PBS as a control. Twenty four hours later, when the swelling was appeared at the injection site, the infected BALB/c mice were injected intravenously with 200 μl of gentamicin–GNPs conjugate at the concentration of 2.7 mg/g GNPs via the tail vein. At 10, 20, 40 and 60 min post‐injection, the mice were sacrificed using CO2 gas. The liver, heart, kidney, small and large intestines, stomach, blood and right and left thigh muscles (normal and infected) were dissected and weighed. The tissues were lysed in sulphuric acid at 600–630°C. Then, the produced ash was dissolved in a mixture of concentrated hydrochloric and nitric acid. The solution was incubated at the room temperature to be dried, a necessary amount of 0.5 N hydrochloric acid was added, and the prepared sample was monitored for gold by an atomic absorption spectrometer (AA240FS, Varian, USA) [8, 19]. For comparison study, 200 μl of free GNPs (2.7 mg/g) was injected intravenously into the tail vein of four groups of five infected BALB/c mice and the in vivo pharmacokinetic study was performed at 10, 20, 40 and 60 min post‐injection in the same way described above. The results were calculated as the percentage of injected dose per gram of each organ (%ID/g). All the animal experiments were approved by Animal Care Committee of Tarbiat Modares University, Tehran, Iran.

2.7 X‐ray imaging

The left lateral thigh muscles of five BALB/c mice were infected in the same way described above. The right thigh muscle of each mouse was injected with PBS as a control. Twenty four hours later, when the swelling was appeared at the injection site, the mice were injected intravenously with 200 μl of gentamicin–GNPs conjugate at the concentration of 2.7 mg/g GNPs via the tail vein. The animals were anesthetised with a combination of xylazine hydrochloride and ketamine hydrochloride prior to X‐ray imaging. The imaging was performed at 10, 20, 40 and 60 min post‐injection of the conjugate using a clinical mammography unit (company PAYAMED, model MAMMO X‐ray unit 100 kHz) with 10 mA s, 0.4 s and 21 kVp.

For comparison study, 200 μl of free GNPs (2.7 mg/g) was injected intravenously into the tail vein of five infected BALB/c mice and X‐ray imaging was performed at 10, 20, 40 and 60 min post‐injection in the same way described above [22, 23].

2.8 Statistical analysis

All the experiments were repeated five times and the results were expressed as mean±standard deviation (SD). ANOVA test was used to compare the biodistribution data of gentamicin–GNPs conjugate and free GNPs in mouse model (p ‐value <0.05).

3 Results

3.1 Synthesis and characterisation of GNPs

According to the UV–vis absorbance spectrum, the synthesised NPs had a strong wavelength band around 520 nm that is typical for gold nanospheres (Fig. 1). The concentration of GNPs was determined 4.96 mg/l. The TEM micrograph (inset image) approved the production of GNPs in spherical shape in the size range of 10–15 nm.

Fig. 1.

Fig. 1

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UV–vis spectrum for synthesised GNPs. Inset image: TEM micrograph of synthesised GNPs with spherical shape in the size range of 10–15 nm

3.2 Characterisation of gentamicin–GNRs conjugate

3.2.1 UV–vis spectroscopy

The intensity of absorption bands at 245 and 520 nm (related to gentamicin and GNPs, respectively) were decreased after the conjugation reaction and accompanied by a new peak emerging at 660 nm (Fig. 2). The appearance of a new peak at 660 nm was due to the replacement of citrate groups by aminoglycosidic antibiotic that was led to formation of gentamicin–GNPs conjugate.

Fig. 2.

Fig. 2

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UV–vis spectra of

a Gentamicin

b GNPs

c Gentamicin–GNPs conjugate.

The appearance of a new peak at 660 nm is due to the conjugation of GNPs with gentamicin

3.2.2 FT‐IR spectroscopy

NH2 stretching frequency of the amino groups at 3423 cm−1 was shifted to the higher wavelength at 3430 cm−1 after the conjugation reaction. This pattern means direct binding of nitrogen atoms of free amino groups of gentamicin molecules to GNP (Fig. 3).

Fig. 3.

Fig. 3

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Infrared spectra of

a Gentamicin

b Gentamicin–GNPs conjugate.

Shifting of NH2 stretching frequency to the higher wavelength means direct binding of nitrogen atoms of free amino groups of gentamicin molecules to GNPs

3.3 Stability in human blood

Fig. 4 depicts the light absorption of gentamicin–GNPs conjugate as a function of wavelength at 6 h after being in human blood. The absorption peak remained intact around 660 nm up to 6 h that showed stability of gentamicin–GNPs conjugate in human blood.

Fig. 4.

Fig. 4

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Absorption spectra of supernatant solution before (black squares) and after (thick line) incubating gentamicin–GNPs conjugate for 6 h in human blood. The preserved absorption peak at 660 nm suggests the stability of the conjugate within the blood microenvironment

3.4 Biodistribution study

Fig. 5 shows the biodistribution of gentamicin–GNPs conjugate in the infected BALB/c mice at 10, 20, 40 and 60 min after intravenous administration expressed as percentage of injected dose per gram of each organ (%ID/g). The results showed significant accumulation of gentamicin–GNPs conjugate in the infected muscle in comparison with normal muscle in all the measured times specially at 40 min post‐injection (p ‐value <0.05).

Fig. 5.

Fig. 5

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Biodistribution data of gentamicin–GNPs conjugate in the vital organs of the infected mice at the time intervals of 10, 20, 40 and 60 min post‐injection. This figure shows significant accumulation of gentamicin–GNPs in the infected muscles in comparison with normal muscles in all the measured times specially at 40 min post‐injection (n = 5). The results represent the mean ± SD of five independent experiments (p‐value <0.05)

Fig. 6 shows no significant accumulation of free GNPs in infected muscle in comparison with normal muscle in all the measured times. These results further showed the targeting ability of gentamicin–GNPs in comparison with free GNPs.

Fig. 6.

Fig. 6

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Biodistribution data of free GNPs in vital organs of infected mice at time intervals of 10, 20, 40 and 60 min post‐injection. The results showed no considerable accumulation of free GNP at the site of infection in comparison with normal muscles (n = 5). The results represent the mean ± SD of five independent experiments (p‐value <0.05)

3.5 X‐ray imaging

Infected muscles were visualised with clarity in X‐ray images in all the time intervals especially at 40 min post‐injection of gentamicin–GNPs conjugate that was in accordance with the result of in vivo pharmacokinetic study. Fig. 7 a shows the image of a representative infected BALB/c mouse at 40 min post‐injection of gentamicin–GNPs conjugate. The visualisation of infection site (left thigh muscle) can be seen properly in comparison with normal muscle (right thigh muscle). Fig. 7 b shows the image of a representative infected BALB/c mouse at 40 min post‐injection of free GNPs. It can be seen that there is not any significant difference between the infected muscle and normal muscle. The relatively accumulation of free GNPs in infected muscle in comparison with normal muscle might be because of the EPR effect. Fig. 7 c shows the image of an infected BALB/c mouse before any injection. These findings demonstrated the ability of gentamicin–GNPs in visualising the infected muscle in comparison with free GNPs.

Fig. 7.

Fig. 7

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Representative X‐ray images of infected BALB/c mice

a At 40 min post‐injection of gentamicin–GNPs conjugate

b At 40 min post‐injection of free GNPs

c Before any injection.

The infected site can be easily seen at 40 min after injection of the new contrast agent in comparison with normal muscle

4 Discussion

In this study, we developed a simple, rapid and effective method for direct binding of gentamicin to citrate functionalised GNPs to produce a contrast agent for X‐ray imaging of infectious foci due to S. aureus. We chose gentamicin as a suitable molecule for conjugation with GNPs, whereas it is bacteriostatic to many Gram‐positive and Gram‐negative microorganisms [19]. Moreover, gentamicin has high ability for binding to GNPs because of having three NH2 groups in its structure [17, 21]. The main arguments for using NPs in drug delivery are that the stability and functionality of the pharmaceutical molecule is further enhanced by its attachment to the NP, and/or that the resulting NPs can be targeted in a way that a soluble, systemically applied compound cannot [24, 25]. The greater antibacterial effect of the GNP conjugates has been attributed to their ability to bind to and/or penetrate the cell wall and, in doing so they are able to deliver a large number of antibiotic molecules into a highly localised volume [26, 27, 28].

The formation of gentamicin–GNPs conjugate was confirmed by UV–vis spectroscopy, FT‐IR and scanning electron microscope analyses. The binding mechanism is still not quite known although it is suggested that the preparation of gold bioconjugates is based on a kind of non‐covalent interaction, such as electrostatic interaction between amino group of gentamicin and negative surface charge of GNPs [18, 21].

NPs usually have short circulation half‐life due to natural defence mechanism of human body for eliminating them after opsonisation by the mononuclear phagocytic system. Therefore, the particle surfaces need to be modified to be invisible to opsonisation [29].

A hydrophilic polymer such as PEG, with a molecular weight of 1900–5000 Da, is prevalently used for this purpose because it has worthwhile attributes such as low degree of immunogenicity and antigenicity, chemical inertness of the polymer backbone, and availability of the terminal primary hydroxyl groups for derivatisation [29, 30, 31]. The result of stability assessment confirmed that the new nanodrug remained stable and did not aggregate while exposed to blood for 6 h [12]. The high stability of gentamicin–GNPs in human blood up to 6 h is quite enough for X‐ray imaging of infection foci. The rate of NPs clearance from blood is highly dependent on NP size. NPs larger than 150 nm are quickly cleared from blood stream by spleen. In contrast, spleen uptake was found to be insignificant when the particles are smaller than 60 nm [12, 18]. Therefore, we chose GNPs in the size range of 10–15 nm in this research. The in vivo pharmacokinetic study showed the ability of gentamicin–GNPs complex in discrimination between normal (right thigh muscles) and infected muscles (left thigh muscles). The conjugate accumulation in the infected muscle in relation to normal muscle at 40 min post‐injection showed the ratio of approximately two times. The results also indicated low retention of the conjugate in liver (1.42 ± 0.21) and its elimination by kidney (12.42 ± 1.3) that are good characteristics for gentamicin–GNPs conjugate as an ideal contrast agent in X‐ray imaging. The localisation of free GNPs in the infected muscle (2.2 ± 0.28) in comparison with gentamicin–GNPs (4.6 ± 0.32) at 40 min post‐injection was not considerable that approves the ability of new contrast agent for targeting the infection site.

X‐ray images confirmed the results of pharmacokinetic study. Using gentamicin–GNPs conjugate as a contrast agent, the infected muscle was visualised in all the measured times specially at 40 min post‐injection in comparison with normal muscle. In contrast, free GNPs were distributed in whole body of mouse model and there was no significant difference between the infected muscle and normal muscle in X‐ray images. This finding again showed the ability of gentamicin–GNPs conjugate as a selective contrast agent in comparison with free GNPs.

5 Conclusion

We successfully synthesised and characterised gentamicin–GNPs conjugate. The results showed that the new conjugate is potentially a promising contrast agent for X‐ray imaging of infection foci in humans which needs future investigations.

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