In Vitro Structural and Functional Evaluation of Gold Nanoparticles Conjugated Antibiotics
© to the authors 2007
Received: 26 June 2007
Accepted: 30 October 2007
Published: 17 November 2007
Bactericidal efficacy of gold nanoparticles conjugated with ampicillin, streptomycin and kanamycin were evaluated. Gold nanoparticles (Gnps) were conjugated with the antibiotics during the synthesis of nanoparticles utilizing the combined reducing property of antibiotics and sodium borohydride. The conjugation of nanoparticles was confirmed by dynamic light scattering (DLS) and electron microscopic (EM) studies. Such Gnps conjugated antibiotics showed greater bactericidal activity in standard agar well diffusion assay. The minimal inhibitory concentration (MIC) values of all the three antibiotics along with their Gnps conjugated forms were determined in three bacterial strains,Escherichia coli DH5α,Micrococcus luteus andStaphylococcus aureus. Among them, streptomycin and kanamycin showed significant reduction in MIC values in their Gnps conjugated form whereas; Gnps conjugated ampicillin showed slight decrement in the MIC value compared to its free form. On the other hand, all of them showed more heat stability in their Gnps conjugated forms. Thus, our findings indicated that Gnps conjugated antibiotics are more efficient and might have significant therapeutic implications.
KeywordsGold nanoparticles Antibiotics Dynamic light scattering Transmission electron microscope Scanning electron microscope Minimal inhibitory concentration Agar well diffusion
Nanotechnology is a rapidly developing field of new therapeutic and diagnostic concept in all areas of medicine [1–3]. Due to their unique characteristics, nanoparticles are considered to have wide applications in detection of biomolecules, drug delivery and release. Of them, Gnps have already been used to deliver protein-based drugs, and are of particular utility because the particles can carry multiple active groups [4–6]. The chemical, optical and electronic properties of Gnps made them well suited for applications in biosensing and therapeutic delivery. Gnps based biosensors [7, 8], drug delivery [9–11] was demonstrated to be more sensitive and effective.
Moreover, nanoparticles were shown to take up by phagocytic cells and held promises as carrier for the treatment of intracellular infections with several antibiotics . It was reported that Gnps as drug carriers allow increased drug concentration at infected sites as well as reduce toxicity of the drug . Thus, Gnps as carrier for the antibacterial drug ciprofloxacin and subsequent release of the drug over an extended period of time was observed . This is essential for ideal antibiotic therapy. Nano carriers were also found to be more effective for the drugs like gentamycin , tuberculosis drugs [16, 17], ampicillin [18–20], anticancer drugs [21, 22], anti fungal drug amphotericin B  etc.
For successful application of nano-antibiotic conjugation, apart from better delivery, their activities should be evaluated properly because the amount of antibiotics often given for therapy is much more higher than the dose required for killing the pathogens. This in turn could produces toxic effect, which was demonstrated in several reports too [24, 25]. For a successful antibiotic therapy, the dose should be reduced to avoid their side effects at the same time the stability should be increased to make them more economic. With the advancement of nanotechnology, functionalized nanoparticles have been used to conjugate different drugs. Among the different nanoparticles, Gnps were found to be less toxic and hence widely used for this purpose. In most of the cases, the conjugation was done by functionalized gold particles, where amino acids, glutathione, polyethylene glycol etc were used as functionalizing agents . But to avoid the possible effects of these agents on biological system, we have conjugated antibiotics directly without any functionalizing agents at the time of the Gnps synthesis .
While there were many reports about the delivery of different drugs in nanoparticles conjugated form, little or no efforts were made, so far, to determine the efficiency, stability of antibiotics conjugated with Gnps in vitro. In this study, we compared the efficiency and stability of Gnps conjugated antibiotics with respect to their free forms in vitro. We found that the MIC of Gnps conjugated ampicillin, streptomycin and kanamycin onEscherichia coli DH5α(Microbial type culture collection (MTCC) No.1652, India),Micrococcus luteus(MTCC No. 106) andStaphylococcus aureus(MTCC No. 96) were reduced when compared to their respective unconjugated free forms. Moreover, the activity of all Gnps conjugated antibiotics showed higher stability compared to their corresponding free forms. Thus our results suggest that antibiotics conjugated with Gnps might be used in therapy for their greater efficiency and stability.
Preparation of Bare Gold Nanoparticles
Gold nanoparticles (Gnps) were prepared by the reduction of chloroauric acid (H[AuCl4) by sodium borohydride. The normal reduction process was performed according to the standard protocol . The size of Gnps obtained by this process was 14 nm.
Preparation of Conjugated Gold Nanoparticles using Antibiotics as Template
Dynamic Light Scattering (DLS)
The Nano-ZS (Malvern) instrument (5 mW HeNe laser λ = 632 nm) was used for this purpose. The sample was taken in a DTS0112—low volume disposable sizing cuvette of 1.5 ml volume (path length 1 cm). The operating procedure was programmed (using the DTS software supplied with the instrument) such that there were average of 25 runs, each run being averaged for 15 s, with an equilibration time of 3 min at 25°C. A particular hydrodynamic diameter (d h) was evaluated several times and the result was presented in terms of distribution of d h .
Transmission Electron Microscopy
All the three Gnps conjugated antibiotics along with the free Gnps were prepared after drying on carbon coated copper grid and observed under a transmission electron microscope (FEI, Model: STWIN) with an accelerating potential of 200 KV and analyzed with TECNAI G2software.
Scanning Electron Microscopy
Gnps conjugated antibiotics along with bare Gnps were lyophilized on glass slides and then coated with gold. The samples were then observed under a scanning electron microscope (JEOL JSM 5200).
MIC Study of Free and Gnps Conjugated Antibiotics
MIC of ampicillin, streptomycin and kanamycin along with their respective Gnps conjugated forms against E. coli DH5 α, M. luteus and S. aureus in Luria-Bertani (LB) broth were determined by standard method . Each tube contained 5 ml of LB medium inoculated with 106 bacteria per ml. Decreasing concentrations of each antibiotic and their corresponding Gnps conjugated form were added to the respective tubes. After 16 h. the turbidity of each tube was measured at 600 nm using a spectrophotometer.
Bactericidal Activity Measurement
This assay was conducted by standard agar well diffusion method. The E. coli DH5 α, M. luteus and S. aureus strains were grown on LB Broth at 37°C overnight upto a turbidity of 0.5 Mac Farland standard (108 CFU per ml) . About 100 μl of this suspension was used to inoculate 90 mm diameter petridish filled with 35 ml of LB agar. Wells (diameter2 = 0.563 cm2) were punched in the agar plates and filled with 100 μl of either antibiotics or their respective Gnps conjugated forms. The concentrations of both the forms of antibiotics were at their respective MIC values, generally used in common laboratory purpose (50 μg/ml for ampicillin, 10 μg/ml for streptomycin and 50 μg/ml for kanamycin) . Plates were incubated at 37°C for overnight. Antibacterial activities were evaluated by measuring the area of zone of inhibition (diameter2). We used autoclaved water and only Gnps as negative control.
Conjugated with Antibiotics
The antibiotics (Fig.1) were conjugated with Gnps by reducing H[AuCl4] with the combined reducing effect of both antibiotics and sodium borohydride. The antibiotics themselves were able to reduce H[AuCl4] to synthesize the Gnps but the reducing power was much less. It took around 4 h for ampicillin and 24 h for streptomycin and kanamycin to reduce H[AuCl4] to form Gnps conjugated nanoparticles (data not shown). Also, in case of ampicillin, the particles produced in this way formed larger aggregates and precipitated out from the solution quickly whereas streptomycin and kanamycin reduced H[AuCl4] very poorly. But the Gnps produced by using the combined reducing property of both sodium borohydride and the antibiotics showed much higher stability. The produced Gnps conjugated antibiotics appeared more bluish (Fig. 3). So, it was obvious that the size of the particles would be larger and that was reflected in the intensity distribution of the size of the Gnps (Fig. 2b). The intensity distribution was obtained due to the Rayleigh scattering (i.e., proportional toR 6, whereR is the radius of particle). We found that there were distributions of large and small particles but the number distribution showed (∼R) that there were major numbers of particle, which have the hydrodynamic radius less than 10 nm (Fig. 2a). The colour showed bluish because of the presence of some larger particles too.
Represents the zone of inhibition (in terms of diameter square) for free antibiotics and antibiotics conjugated with Gnps in three bacterial strains
Name of the bacterial strain
Name of antibiotics
Inhibitory zone in sq. diameter (cm2)
% Change in inhibitory sq. diameter
E. coli DH5 α (Gram −Ve)
3.085 ± 0.146
3.569 ± 0.160
2.189 ± 0.057
2.453 ± 0.102
3.371 ± 0.164
4.545 ± 0.223
M. luteus(Gram +Ve)
8.740 ± 0.201
10.493 ± 0.354
0.818 ± 0.091
1.712 ± 0.241
2.507 ± 0.118
2.960 ± 0.149
S. aureus(Gram +Ve)
14.839 ± 0.321
16.659 ± 0.678
1.588 ± 0.098
2.132 ± 0.150
Represents minimal inhibitory concentrations (MIC) for free antibiotics along with their respective Gnps conjugated form in three bacterial strains
Name of the bacterial strain
Name of antibiotics
Minimal inhibitory concentration (μg/ml) for 106 bacteria/ml
% Change in MIC
E. coli DH5α(Gram −Ve)
50.0 ± 0.50
45.0 ± 1.50
14.0 ± 2.00
7.0 ± 1.00
30.0 ± 2.50
12.0 ± 1.00
M. luteus(Gram +Ve)
0.52 ± 0.02
0.45 ± 0.03
22.0 ± 2.00
17.0 ± 1.00
32.5 ± 0.50
23.0 ± 1.50
S. aureus(Gram +Ve)
0.45 ± 0.03
0.37 ± 0.01
9.0 ± 0.50
5.8 ± 0.20
Represents the bactericidal activity inE. coli DH5 α by agar well diffusion assay for free antibiotics and their respective Gnps conjugated form after different temperature and time stresses
Zone of inhibition forE. coli DH5 α strain in sq. cm
Incubated for 10 min at
Storage at room temp. (25–28°C) for
Our results for the first time demonstrated that the in vitro bactericidal activity of Gnps conjugated ampicillin, streptomycin and kanamycin were more efficient compared to their respective free forms. We had also developed a simple technique for the conjugation of antibiotics with Gnps during its synthesis step. Usually, such conjugation needs functionalization process. But we avoided the interference of such functionalizing agent in determining the bactericidal activity of the antibiotics. Using the combined reducing property of antibiotics and borohydride, antibiotics were conjugated with Gnps. The interaction between antibiotics and Gnps is likely to be mediated by the adsorption of the antibiotic molecules on the nanoparticle surfaces. The average particles size after conjugation were shown to decrease (Fig. 2a). This was possibly again due to the combined reducing property of both antibiotics and borohydride in situ. However, the plasmon resonance study (Fig. 3) showed a red shift, indicating the presence of larger particles (Fig. 2b), though they were less in number (Fig. 2a). In case of Gnps conjugated ampicillin, the plasmon resonance showed a flatten plateau in the plasmon region due to the presence of such poly dispersed particles. The dynamic light scattering study (Fig. 2b) and TEM study (Fig. 4b) also supported the above statement. In one step further, we directly showed evidences by scanning electron microscopic studies that, all the three antibiotics formed some specific three-dimensional structures when conjugated with Gnps. Also, to prove the conjugation of antibiotics with Gnps, we found that after spinning down the Gnps conjugated antibiotics, the functional activity of the precipitate (pellet-suspension) was about 60–80% and that of the supernatant was about 20–40%. Thus, majority of the antibiotic molecules were associated with Gnps.
Using standard agar well diffusion assay, we compared the bactericidal activity of Gnps conjugated antibiotics with their respective free forms. The relative bactericidal activity of Gnps conjugated ampicillin was less effective than Gnps conjugated streptomycin and kanamycin (Fig. 7 and Table 1). Consequently, for E. coli DH5 α strain, the MIC values of Gnps conjugated ampicillin decreased 10%, while the percentage decrement for Gnps conjugated streptomycin and kanamycin were 50% and 60%, respectively. Such differential activity might be due to the differences in the mode of action of the antibiotics. Ampicillin inhibits the cell wall biosynthesis by inhibiting the cross-linking reaction mediated by transpeptidase, while both streptomycin and kanamycin bind with ribosome and block translation process during protein synthesis . The binding affinity of Gnps conjugated antibiotics with the said enzyme or even ribosome might be the key factor for this differential response. Although, in the control experiments, only Gnps did not show any bactericidal activity (Fig. 6) so the antibiotics conjugated with Gnps might have a higher binding affinity to their respective targets. On the other hand, the Gnps conjugated antibiotics might have greater chance to penetrate bacterial cell membrane compared to their respective free forms. In the control experiments, we also mixed pre-synthesized Gnps and antibiotics externally to determine the bactericidal activity. None of these antibiotics mixed with Gnps showed significant increment in bactericidal activity compared to the respective free antibiotics (data not shown). Thus, only Gnps did not promote the penetration of the antibiotics into the bacterial cells. So, Gnps conjugated antibiotics might have some other mechanisms that could enhance the efficacy of the antibiotics. On the other hand, presence of sodium borohydride during the Gnps synthesis step might alter the function of antibiotics, but when we mixed only sodium borohydride with antibiotics, the functional activity of antibiotics did not increase much (only 3–6%). Thus the reduction process in our reaction condition does not change the antibiotic structure abruptly. The conjugation between the antibiotics and Gnps is probably based on the adsorption phenomenon mediated by intermolecular forces. Thus having the larger surface area of these adsorbed antibiotics in Gnps conjugated form, their bactericidal activity might increases compared to their respective free forms. However, the exact mechanisms of action of Gnps conjugated antibiotics are highly speculative and needs further study.
The Gnps conjugated antibiotics were seen to be more stable than their respective free forms. Stability of the most antibiotics is temperature and parenteral solutions dependent . We introduced stresses by heat shock and by prolong storage at room temperature (25–28°C). In both the cases Gnps conjugated antibiotics were observed to be more stable compared to their respective free forms except Gnps conjugated ampicillin during its temporal study. This was perhaps due to the close association between Gnps and antibiotics, the bond energy of antibiotic molecules were increased which in turn stabilized them. Whatever the mechanisms of such stability of Gnps conjugated antibiotics be, we showed further that at elevated temperature the Gnps conjugated forms were even more active for ampicillin. This was perhaps due to the delocalization of the electron in the carbonyl group of the β-lactam ring in ampicillin at elevated temperature. Elevation in temperature might induce breakage in the β-lactam ring of the free ampicillin, whereas Gnps conjugation might stabilize the ring and thereby allowing the delocalization of electron. Hence, in case of free ampicillin we found a decrease in the activity, whereas Gnps conjugated form showed more activity than the activity at its lower temperature. In this regard, one of the important findings was the deactivation of streptomycin at 90°C, whereas its Gnps conjugated form retained its activity at the same condition. Here also, the Gnps conjugation might stabilize the structure of the streptomycin molecules.
We found that the activity of Gnps conjugated ampicillin decreased compared to its free form after two weeks (Table 3). Actually, we observed that the Gnps conjugated ampicillin (Table 3) was precipitated out from the solution. This might be the reason for its decreased efficiency compared to its free form.
It was reported that antibiotic solutions used for longer than 7 days should be stored at 4°C, those stored at 24°C should be discarded after 7 days . Our data also supported this observation. Moreover, we provided evidences that Gnps conjugated antibiotics were more stable and might withstand more harsh storage conditions, which raised a hope to use Gnps conjugated antibiotics with greater efficiency in the remote area, where proper storage condition is unavailable.
Biswarup Saha and Jaydeep Bhattacharya authors contributed equally.
We thank Dr. Joydeep Mukherjee, School of Environmental Sciences, Jadavpur University, Kolkata, for the gift of bacterial strains,Micrococcus luteus andStaphylococcus aureus. We thank Mr. Pallab Dasgupta, Department of Instrumentation Science, Jadavpur University, Kolkata for helping us to avail the SEM facility. We also acknowledge Dr. Pulak Ray and Mr. Tapan Kumar Ray of TEM Section, Saha Institute of Nuclear Physics, Kolkata, for providing the TEM facilities. This work was partially supported by CSIR, India (financial grant No. 37(1231)/02/EMR-II).
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