Doxycycline conjugated with polyvinylpyrrolidone-encapsulated silver nanoparticles: a polymer's malevolent touch against Escherichia coli

Abstract

The emergence of multi-resistant pathogens has necessitated the investigation of new strategies to cope with this ever-increasing threat to public health. In this context, we combined silver nanoparticles (AgNPs) with doxycycline (DO), an antibiotic from the class of tetracyclines, to evaluate the potentiality of this hybrid as a bactericidal agent against E. coli. Polyvinylpyrrolidone (PVP) was used as a stabilizer to prevent the excessive growth and agglomeration of AgNPs. Interestingly, DO bound directly to PVP and had its concentration increased around the particle as a consequence of this interaction. As a result, the AgNPs/DO conjugates presented enhanced bactericidal properties compared to the individual components. Stabilizing agents are generally unwanted on the surfaces of nanoparticles because of their potential to block adsorption surface sites. However, we have shown that PVP played a paramount role in concentrating DO around the particle, which culminated in an increased bactericidal activity towards E. coli.

---

1. Introduction

Silver has long been known to prevent antimicrobial activity,^1–4 but the advent of antibiotics has severely reduced its use for bactericidal purposes. The ability of various microorganisms to adapt to antimicrobial drugs and the improper administration of antibiotics have enabled emergence of multi-drug-resistant pathogens,^5,6 for which treatment with broad-spectrum antibiotics is less effective, more toxic, and expensive. This has led to the renaissance of silver-based bactericidal agents, especially in the nanoregime,^7–10 which display considerable broad-spectrum antimicrobial activity. An interesting approach is the combination of nanoparticles and antibiotics to yield a more potent antimicrobial agent.^11,12 Due to their large surface-area-to-volume ratio and biocompatibility, inorganic nanoparticles are considered to be ideal candidates for carrying large amounts of antibiotics without compromising their activity. Li et al.¹³ showed that the combination of silver nanoparticles with amoxicillin resulted in greater bactericidal effect on Escherichia coli than when the components are administered separately. Rai et al.¹⁴ used cefaclor, a second-generation antibiotic, as both reducing and capping agent to produce spherical gold nanoparticles. The authors showed that the nanogold–cefaclor hybrid exhibited a potent antimicrobial activity against both Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria. The conjugation of antibiotics with inorganic nanoparticles might be a strategy against multidrug-resistant bacteria.

In this study, we showed that the combination of polyvinylpyrrolidone (PVP)-capped silver nanoparticles (AgNPs) with doxycycline (DO), an antibiotic from the class of tetracyclines, yields a hybrid agent for the inhibition of E. coli. The results showed that PVP had a key role in linking DO to the nanoparticle, thus the system profits from the combination of the intrinsic bactericidal properties of silver with the increased concentration of DO around the particle. To the best of our knowledge, this is the first report on the combination of DO with a polymer-coated silver nanoparticle. This process might be suitable for coating surgical instruments, implants, and other surfaces that require hygienic conditions.

2. Experimental section

2.1. Chemicals and reagents

Silver nitrate, polyvinylpyrrolidone (PVP; molecular weight = 10 000), sodium hydroxide, glycerol and DO hyclate (>98%) were obtained from Sigma-Aldrich Chemical Co. Escherichia coli strain INCQS 00171 was cultivated in our laboratory.

2.2. Production and characterization of AgNPs

AgNPs were produced using a previously reported method.^15,16 Briefly, all glassware was kept overnight in a KMnO₄ + NaOH solution, rinsed with deionized water, kept for 10 min in an H₂O₂ + H₂SO₄ solution (1 : 1 v/v), again rinsed with deionized water and dried prior to use. The following stock solutions were prepared: 50 mmol L⁻¹ of AgNO₃, 166 g L⁻¹ of PVP, and a solution containing 1.0 mol L⁻¹ of NaOH + 1.0 mol L⁻¹ of glycerol. Known volumes of PVP and AgNO₃ solutions were dissolved in water to yield a 5 mL solution. In a separate beaker, a determined volume of NaOH + glycerol solution was mixed in water to generate a 5 mL solution. The glycerol–NaOH solution was then added to that of the AgNO₃–PVP to yield the final concentrations displayed in Table 1, in which the coded and real values for the 2² full-factorial design, conceived with the help of the software MODDE® 4.0 (Umetrics, Umeå, Sweden), can be observed. The pH values of the AgNPs colloidal solutions were then adjusted to 7 by the addition of diluted HCl.

Table 1 Full factorial experimental design layout

Runs	Coded levels		Real values		Response
	x1	x2	Ag^+ (mmol L^−1)	PVP (g L^−1)	Size/nm
1	+1	−1	1.0	0.50	38.2 ± 0.9
2	+1	+1	1.0	5.0	27.2 ± 0.8
3	+1	+1	1.0	5.0	26.0 ± 0.9
4	−1	+1	0.010	5.0	72.3 ± 2.2
5	+1	−1	1.0	0.50	39.2 ± 0.6
6	−1	+1	0.010	5.0	78.0 ± 5.4
7	−1	−1	0.010	0.50	96.6 ± 2.1
8	−1	−1	0.010	5.0	80.0 ± 3.6
9	+1	−1	1.0	0.50	37.3 ± 2.1
10	+1	+1	1.0	5.0	22.3 ± 0.6
11	+1	+1	1.0	5.0	24.3 ± 0.8
12	−1	+1	0.010	5.0	80.3 ± 7.9
13	+1	−1	1.0	0.50	37.5 ± 2.0
14	−1	+1	0.010	5.0	57.1 ± 3.9
15	−1	−1	0.010	0.50	77.7 ± 5.4
16	−1	−1	0.010	0.50	85.2 ± 2.8

---

UV-vis absorption spectra of the AgNPs were acquired with an Evolution 60S UV-visible spectrophotometer (Thermo Scientific). Dynamic light scattering (DLS) and fluorescence spectroscopy were carried out with a ZetaPlus instrument and a PerkinElmer LS45 fluorometer, respectively. Small-angle X-ray scattering (SAXS) was conducted on the D1B beamline at the Brazilian Synchrotron Light Laboratory (LNLS). For this experiment, liquid samples (with water as the solvent) were injected into an in-vacuum cell with parallel mica windows at a sample-to-detector distance of 1500 mm. The monochromatized X-ray beam had a wavelength of 1.488 nm, and the detector was a Pilatus 300k. FTIR in the ATR mode was performed with a Bruker Vertex 70 spectrophotometer.

2.3. Conjugation of DO with AgNPs

The conjugation of AgNPs with DO was achieved by simple incubation. Various volumes of a 10 mmol L⁻¹ doxycycline stock solution were added to the AgNPs colloidal solutions to produce distinct concentrations of the antibiotic. All the above-mentioned techniques were used to characterize the AgNPs–antibiotic complex.

2.4. Bacteriological experiments

E. coli (INCQS 00171) was cultured in a Müller-Hinton agar medium on a Petri dish for 24 h at 37 °C. Bacterial colonies were then transferred into a 0.9% saline solution to yield a suspension with a turbidity of 0.5 MacFarland, which is equivalent to 1 × 10⁸ UFC mL⁻¹. The latter suspension was then swabbed onto another Petri dish containing the Müller-Hinton agar medium. Subsequently, with the help of a sterilized stainless-steel cylinder, 6 mm wells were made in the culture medium and filled with 50 μL of antimicrobial agents (DO, AgNPs, and DO conjugated with AgNPs). The plates were incubated for 24 h at 37 °C and their inhibition zones were recorded and expressed in millimeters.

3. Results and discussion

3.1. Synthesis and characterization of AgNPs

In this study, AgNPs were synthesized by reducing Ag⁺ with glycerol in an alkaline medium at room temperature. The current production of glycerol is sufficient for its main use as a raw material in the pharmaceutical and cosmetics industries. Because glycerol is now also produced as a byproduct of biodiesel fabrication, the availability of this alcohol has surpassed its current demand, making it a relatively inexpensive chemical.¹⁷ Due to its biodegradability under aerobic conditions, non-toxicity, and low price, glycerol is a more attractive option for the generation of nanoparticles when compared to current reducing chemicals such as formamide, sodium borohydride and hydrazine. Based on preliminary UV-vis results from our group, glycerol alone is not capable of acting concomitantly as both reducing and stabilizing agent, thus the use of a capping agent is imperative to prevent aggregation and precipitation of AgNPs. PVP has been selected for the task due to its intrinsic non-toxicity¹⁸ and capping properties.^15,16,19 Table 1 shows the coded levels and real values with their effects on the size of the AgNPs. As the results were acquired with DLS, the hydrodynamic diameter of the particles is indeed investigated, meaning that the measured size takes into account the adsorbed PVP. Table 1 shows that large nanoparticles (96.6 nm ± 2.1 nm) are obtained at the lowest PVP and Ag⁺ concentrations and a possible agglomeration cannot be ruled out either. In contrast, the highest values of the variables generated smaller nanoparticles with narrower size distribution (22.3 nm ± 0.6). These results are summarized in Fig. 1, in which one can note that both factors, PVP and Ag⁺, are relevant for the determination of the size of the nanoparticles; however, Ag⁺ concentration was found to deliver the most pronounced effect on the size of the AgNPs. This is probably due to the autocatalytic nature of the formation process of AgNPs.²⁰ The AgNPs initially pass through a latency period, the length of which depends on Ag⁺ concentration. At high Ag⁺ concentrations, many nuclei are rapidly formed which, in turn, speed up the reaction because they serve as seeds for the reaction Ag⁺ → Ag⁰. At low Ag⁺ concentrations, the process is generally slower, thus silver is preferable for incorporation into the few formed nuclei, which leads to larger particles.

Fig. 1 Effect of Ag⁺ and PVP concentrations on the sizes of AgNPs. Results were acquired with DLS. +1 and −1 represent the highest and lowest concentration values, respectively, of Ag⁺ and PVP.

pH was kept at 13 because alkaline media are imperative for the synthesis of silver nanoparticles, as previously shown for silver¹⁵ and gold.²¹ Fig. 2A reveals the effect of PVP concentration on the UV-vis spectra of the AgNPs, with the silver concentration kept at 1 mmol L⁻¹. For the PVP concentration of 5.0 g L⁻¹ [PVP(+)], the spectrum is centered at 400 nm, a value that corresponds to the characteristic surface plasmon resonance (SPR) of PVP-stabilized AgNPs¹⁵ in the same chemical environment. The absence of multiple bands is indicative of isotropic AgNPs,²² which will be confirmed later by TEM. In addition, the symmetry of the band suggests a low degree of aggregation in the solution.²³ The lowest concentration of PVP [PVP(−)] caused a slight redshift of the maximum wavelength to 407 nm, suggesting an overall particle size increase.²⁴ Fig. 2B depicts the impact of Ag⁺ concentration on the shape of the UV-vis spectra of AgNPs with PVP kept at its highest value. At low Ag⁺ concentrations [Ag⁺(−)], the spectrum is redshifted compared to that from high Ag⁺ concentration, thus corroborating the results from DLS, which show a particle size increase.

Fig. 2 Effect of (A) PVP concentration and (B) Ag⁺ concentration on UV-vis spectra of the AgNPs. (C) TEM images with respective size distributions.

All the AgNPs samples were analyzed with TEM, and the results are shown in Fig. 2C. The size distribution of the Ag(+)PVP(+) sample clearly presents a bimodal feature, with the first and second populations having average sizes of 2.9 nm ± 1.0 nm and 15.1 nm ± 5.1 nm, respectively. A bimodal size distribution stems from the dissolution of small particles and redeposition of the dissolved species onto the surface of larger particles, hence spawning two particle populations. It is important to indicate that more than 70% of the nanoparticles are comprised in the population centered at 2.9 nm, as seen in the respective histogram. Once the average particle size increases, the bimodal feature tends to disappear, as in the case of the Ag(+)PVP(−) sample, in which the average size distribution is 4.0 nm (±1.8 nm). The size distribution broadened slightly as a consequence of low PVP amount, which is corroborated by UV-vis spectroscopy. The size of AgNPs substantially increased when Ag⁺ concentration was set at its lowest value. The Ag(−)PVP(+) and Ag(−)PVP(−) presented average sizes of 24 nm ± 8.0 nm and 18.0 ± 22 nm, respectively. In the case of the latter (at the lowest PVP amount), the standard deviation was substantially larger than that of the former due to the low PVP concentration, which led to a poor capping capacity. This is consistent with the results shown in Fig. 1, which demonstrated that the lowest concentrations of both Ag⁺ and PVP generated larger nanoparticles with broader size distributions. It is interesting to notice that DLS significantly overestimates the size of the nanoparticles because it measures the hydrodynamic radius, which takes into account the layer of adsorbed PVP.

3.2. Conjugation of DO with AgNPs

The next step comprises the conjugation of the AgNPs with DO, which in principle can be monitored via the quenching of the AgNPs fluorescence signal. Fig. 3A shows the fluorescence emission spectra of AgNPs at distinct concentrations of DO. It can be observed that the lowest concentration of DO (16 μmol L⁻¹) is sufficient to cause some fluorescence quenching; moreover, at the concentration of 144 μmol L⁻¹, the fluorescence signal is practically suppressed. Fig. 3B presents the fluorescence intensity before the addition of DO (F₀) over the fluorescence intensity after the addition of DO (F). The plot deviates from the classic Stern–Volmer behavior, which suggests a non-linear DO–AgNPs interaction.²⁵ A somewhat linear behavior is observed only at low DO concentrations. Non-linear Stern–Volmer plots are often found for macromolecules²⁶ and nanoparticles²⁷ and are normally attributed to static quenching.²⁸ However, in our case, we may have to consider the possibility that the DO is simply within the sphere of action of the fluorophore; a situation in which the quencher (DO) is close enough to the fluorophore at the moment of excitation and instantly quenches the excited state.²⁹ This hypothesis is supported by the existence of a capping polymer that might prevent DO from directly binding to the AgNPs. To shed light into this matter, fluorescence lifetime experiments were conducted for AgNPs in the absence and presence of DO, and the results are shown in the inset of Fig. 3C. Analysis of the plots essentially gave the same lifetimes of 6.90 ns and 7.25 ns for AgNPs and AgNPs mixed with DO, respectively, suggesting that DO is not adsorbed directly onto the AgNPs. Nevertheless, DO is being accumulated near the AgNPs, as depicted by Fig. 3B. A plausible explanation would be that the capping polymer PVP interacts with DO, causing its accumulation at the nanoparticle's surface.

Fig. 3 (A) Fluorescence emission spectra of AgNPs (excitation wavelength = 350 nm) at distinct concentrations of doxycycline, (B) fluorescence intensity before the addition of DO (F₀) over the fluorescence intensity after the addition of various doxycycline concentrations (F), and (C) fluorescence lifetime of AgNPs in the presence and absence of doxycycline.

Fig. 4 presents the FTIR spectra in ATR mode of DO, DO mixed with AgNPs, and DO mixed only with PVP. The molecular structure of DO (Scheme 1) is also provided for clarification. As the most characteristic region of the DO spectrum comprises the range of 1200–1800 cm⁻¹, only this region has been analyzed in detail. The bands have been assigned according to the studies of Gu and Karthikeyan³⁰ and Lacher et al.³¹ The bands for DO (black line) were assigned in the following manner: frequencies at 1677 cm⁻¹ and 1537 cm⁻¹ are related to the carbonyl and amino groups of the amide, respectively, in ring A; bands at 1613 cm⁻¹ and 1581 cm⁻¹ correspond to the carbonyl groups in rings A and C, respectively; the band at 1458 cm⁻¹ is related to the C=C skeleton vibration. The oxygen atoms on C1 and C3 may be equivalent because there was only one frequency at 1615 cm⁻¹.³² The spectrum of DO mixed with PVP-capped AgNPs (Fig. 4A, blue line) clearly shows band changes of amide carbonyl and amino groups in ring A. Upon mixing DO with PVP-capped AgNPs, the peaks for amide C=O of DO shifted from 1677 cm⁻¹ to 1670 cm⁻¹ and those of –NH₂ shifted from 1536 cm⁻¹ to 1542 cm⁻¹. These results suggest that the complexation of DO with PVP could take place through the amide C=O in ring A. These shifts have also been observed in the spectrum of DO mixed with PVP alone, which corroborates the idea that DO is interacting mainly with PVP, as depicted in Fig. 4B.

Fig. 4 FTIR spectra in the ATR mode of (A) 10 mmol L⁻¹ doxycycline and 10 mmol L⁻¹ doxycycline mixed with AgNPs and (B) 10 mmol L⁻¹ doxycycline and 10 mmol L⁻¹ doxycycline mixed with 5 g L⁻¹ PVP.

Scheme 1

Another point worth mentioning in Fig. 4A is the increased band signal upon the addition of AgNPs into the DO solution. Because ATR is a technique that probes the interfacial region between the solution and the ATR crystal, more intense signals mean a higher concentration of the species in question at the interface. Due to the ease of baseline illustration, the bands at 1582 cm⁻¹ were chosen to be integrated and compared with that of pure DO, as depicted in Fig. 5. In the latter graphic, it is clearly observed that the addition of PVP-capped AgNPs into the DO solution resulted in an increase in band intensity, which is justified by an increased concentration of DO around the particle promoted by the polymer. This interpretation is in accordance with the fluorescence results presented in Fig. 3B, in which the fluorescence quenching is caused by high concentrations of DO due to PVP–DO interaction. Interestingly, PVP itself is not able to promote a local concentration increase (Fig. 5), which suggests that the structure and concentration of PVP around the nanoparticle may also play a role in DO encapsulation. AgNPs produced with the lowest and highest PVP concentrations promoted 6% and 20% increase in DO concentration around the particles, respectively.

Fig. 5 Effect of the mixing material on the band at 1585 cm⁻¹ for DO.

The UV-vis spectra of DO and DO mixed with PVP-capped AgNPs in Fig. 6 further prove that these species interact with each other. A UV-vis spectrum of DO (red curve) exhibits a maximum at 346 nm, which is shifted to 363 nm upon mixing with AgNPs (blue curve). This implies that the tricarbonylamide group in ring A is involved in complexation with PVP, which is corroborated by the ATR experiments presented in Fig. 4. Further, SAXS measurements revealed that DO has practically no effect on the size of the AgNPs (Fig. 7), as well as on their distribution; this is an important result that can rule out size effects in the bacteriological tests.

Fig. 6 UV-vis spectra of DO, AgNPs, and DO mixed with AgNPs.

Fig. 7 Effect of DO on the size distribution of AgNPs obtained by SAXS.

3.3. Antimicrobial tests

Well diffusion method was adopted to access the antimicrobial potentiality of the AgNPs as well as that of the AgNPs–DO. As shown in Fig. 8, growth of E. coli was inhibited by the conjugates, which is demonstrated by the round, lighter-shaded area around the wells. In these images, the inhibition zone at the centers of the Petri dishes was due to a DO solution used as a control to check the bacterial susceptibility to antibiotics. It is important to stress that the DO concentration of 30 μg mL⁻¹ in the control was the same for all the conjugates.

Fig. 8 Inhibition zones for DO conjugated with (A) Ag(+)PVP(+) (left side) and Ag(+)PVP(−) (right side) and (B) Ag(−)PVP(+) (left side) and Ag(−)PVP(−) (right side).

Fig. 9 summarizes the antibacterial results from the conjugates, and several important features can be promptly realized from it. The components (glycerol and PVP) used to synthesize the nanoparticles did not present any antibacterial effect (results not shown). The AgNPs produced with the highest concentration of Ag⁺ successfully prevented bacterial growth, which means that conjugation-free particles are also efficient for this purpose. At the same loading of silver, the Ag(+)PVP(+) nanoparticles delivered somewhat larger inhibition zones than the Ag(+)PVP(−) probably because the latter are slightly larger than the former. It has been discussed in the literature that small silver nanoparticles are more active than those of larger size due to the larger surface-area-to-volume ratio of the former, which leads to a larger contact area between the nanoparticles and bacteria.³³ Another mechanism is the uptake of free silver ions that are released by the AgNPs, followed by the disruption of ATP production and an increase in DNA mutation.³⁴ Naturally, smaller AgNPs release more silver ions than larger ones, therefore preventing bacterial growth more efficiently. Upon the conjugation of AgNPs with DO, it was observed that the inhibition zones of the conjugates are larger than those of the respective AgNPs alone. It was also expected that the conjugates delivered inhibition zones larger than those calculated by summing the inhibition zones of the components alone (DO + AgNPs). However, an inspection of the results in Fig. 9 shows that this was not the case for the Ag(+)PVP(+) and Ag(+)PVP(−) conjugates. It is well known that tetracyclines form strong complexes with aluminum and iron,³⁰ and a possible complexation between silver ions and DO cannot be ruled out. This could decrease the diffusion of the new species throughout the agar gel, which would in turn spawn smaller inhibition zones within the experimental period. Chin and Lach³⁵ showed that the diffusion rates of tetracycline chelates are reduced even if the chelate is water soluble.

Fig. 9 Bar graph representation of inhibition zones from well diffusion experiments carried out with the AgNPs and their respective DO conjugates. NO means “not observed”. Silver loadings: 8.6 μg for Ag(+)PVP(+), Ag(+)PVP(−), and their conjugates with DO; 0.090 μg for both Ag(−)PVP(+) and its DO conjugate; and 0.084 for both Ag(−)PVP(−) and its DO conjugate.

The most striking results were the antimicrobial activities of the conjugates from nanoparticles produced with the lowest amount of silver ions. It can be observed in Fig. 9 that the non-conjugated Ag(−)PVP(+) and Ag(−)PVP(−) did not produce any inhibition zones probably due to the low concentration of nanoparticles and their large sizes. On the other hand, inhibition zones even larger than that of DO alone were observed for the Ag(−)PVP(+)/DO and Ag(−)PVP(−)/DO conjugates. This may be explained by the ability of the PVP-capped AgNPs to concentrate DO around them, thus locally generating DO concentrations greater than that of the bulk. We have already discussed in Fig. 5 that even AgNPs with low amounts of PVP are capable of concentrating DO. From this result, it is clear that the pivotal role is played by PVP in bonding with DO. As there is now less silver to be released due to the larger size of the AgNPs, DO can diffuse more freely.

4. Conclusions

Herein, we presented a new approach to combine DO with silver nanoparticles. The stabilizing agents are normally unwanted on the surface of nanoparticles because they block potential adsorption surface sites. However, we have shown that PVP played a paramount role in concentrating DO around the particle, which culminated in an increased bactericidal activity towards E. coli.

Acknowledgements

The authors are grateful to CNPq (grant no. 442087/2014-4) and to Prof. Frank H. Quina for the time-resolved fluorescence measurements. M.B.C. and J.F.A.O. would like to thank Fapesp through the project numbers: 2014/22322-2 and 476798/2010-8.

References

1. T. J. Berger, J. A. Spadaro, S. E. Chapin and R. O. Becker, Electrically generated silver ions: quantitative effects on bacterial and mammalian cells, Antimicrob. Agents Chemother., 1976, 9, 357–358.
2. H. Liedberg and T. Lundeberg, Silver alloy coated catheters reduce catheter-associated bacteriuria, Brit. J. Urol., 1990, 65, 379–381.
3. B. Jansen, M. Rinck, P. Wolbring, A. Strohmeier and T. Jahns, In vitro evaluation of the antimicrobial efficacy and biocompatibility of a silver-coated central venous catheter, J. Biomater. Appl., 1994, 9, 55–70.
4. Q. L. Feng, J. Wu, G. Q. Chen, F. Z. Cui, T. N. Kim and J. O. Kim, A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus, J. Biomed. Mater. Res., 2000, 52, 662–668.
5. D. M. Livermore, Introduction: the challenge of multiresistance, Int. J. Antimicrob. Agents, 2007, 29(suppl. 3), S1–S7.
6. G. D. Wright and A. D. Sutherland, New strategies for combating multidrug-resistant bacteria, Trends Mol. Med., 2007, 13, 260–267.
7. Y. Zhang, H. Peng, W. Huang, Y. Zhou and D. Yan, Facile preparation and characterization of highly antimicrobial colloid Ag or Au nanoparticles, J. Colloid Interface Sci., 2008, 325, 371–376.
8. L. Balogh, D. R. Swanson, D. A. Tomalia, G. L. Hagnauer and A. T. McManus, Dendrimer–silver complexes and nanocomposites as antimicrobial agents, Nano Lett., 2001, 1, 18–21.
9. J. F. Hernández-Sierra, F. Ruiz, D. C. Cruz Pena, F. Martínez-Gutiérrez, A. E. Martínez, A. d. J. P. Guillén, H. Tapia-Pérez and G. M. Castañón, The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold, Nanomedicine, 2008, 4, 237–240.
10. J. S. Kim, E. Kuk, K. N. Yu, J.-H. Kim, S. J. Park, H. J. Lee, S. H. Kim, Y. K. Park, Y. H. Park, C.-Y. Hwang, Y.-K. Kim, Y.-S. Lee, D. H. Jeong and M.-H. Cho, Antimicrobial effects of silver nanoparticles, Nanomedicine, 2007, 3, 95–101.
11. M. J. Rosemary, I. MacLaren and T. Pradeep, Investigations of the antibacterial properties of ciprofloxacin@SiO₂, Langmuir, 2006, 22, 10125–10129.
12. R. Jagannathan, P. Poddar and A. Prabhune, Cephalexin-mediated synthesis of quasi-spherical and anisotropic gold nanoparticles and their in situ capping by the antibiotic, J. Phys. Chem. C, 2007, 111, 6933–6938.
13. P. Li, J. Li, C. Wu, Q. Wu and J. Li, Synergistic antibacterial effects of β-lactam antibiotic combined with silver nanoparticles, Nanotechnology, 2005, 16, 1912.
14. A. Rai, A. Prabhune and C. C. Perry, Antibiotic mediated synthesis of gold nanoparticles with potent antimicrobial activity and their application in antimicrobial coatings, J. Mater. Chem., 2010, 20, 6789–6798.
15. A. C. Garcia, L. H. S. Gasparotto, J. F. Gomes and G. Tremiliosi-Filho, Straightforward synthesis of carbon-supported Ag nanoparticles and their application for the oxygen reduction reaction, Electrocatalysis, 2012, 3, 147–152.
16. L. H. S. Gasparotto, A. C. Garcia, J. F. Gomes and G. Tremiliosi-Filho, Electrocatalytic performance of environmentally friendly synthesized gold nanoparticles towards the borohydride electro-oxidation reaction, J. Power Sources, 2012, 218, 73–78.
17. J. F. Gomes, F. B. C. de Paula, L. H. S. Gasparotto and G. Tremiliosi-Filho, The influence of the Pt crystalline surface orientation on the glycerol electro-oxidation in acidic media, Electrochim. Acta, 2012, 76, 88–93.
18. B. Nair, Final report on the safety assessment of polyvinylpyrrolidone (PVP), Int. J. Toxicol., 1998, 17, 95–130.
19. I. Pastoriza-Santos and L. M. Liz-Marzan, Formation of PVP-protected metal nanoparticles in DMF, Langmuir, 2002, 18, 2888.
20. K. Esumi, T. Hosoya, A. Suzuki and K. Torigoe, Formation of gold and silver nanoparticles in aqueous solution of sugar-persubstituted poly(amidoamine) dendrimers, J. Colloid Interface Sci., 2000, 226, 346–352.
21. E. B. Ferreira, J. F. Gomes, G. Tremiliosi-Filho and L. H. S. Gasparotto, One-pot eco-friendly synthesis of gold nanoparticles by glycerol in alkaline medium: role of synthesis parameters on the nanoparticles characteristics, Mater. Res. Bull., 2014, 55, 131–136.
22. G. Zhang, J. Jasinski, J. Howell, D. Patel, D. Stephens and A. Gobin, Tunability and stability of gold nanoparticles obtained from chloroauric acid and sodium thiosulfate reaction, Nanoscale Res. Lett., 2012, 7, 337.
23. N. Vigneshwaran, R. P. Nachane, R. H. Balasubramanya and P. V. Varadarajan, A novel one-pot ‘green’ synthesis of stable silver nanoparticles using soluble starch, Carbohydr. Res., 2006, 341, 2012–2018.
24. M. C. Daniel and D. Astruc, Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev., 2004, 104, 293–346.
25. B. Pan, B. Xing, W. Liu, G. Xing and S. Tao, Investigating interactions of phenanthrene with dissolved organic matter: limitations of Stern–Volmer plot, Chemosphere, 2007, 69, 1555–1562.
26. J. R. Lakowicz and G. Weber, Quenching of fluorescence by oxygen. Probe for structural fluctuations in macromolecules, Biochemistry, 1973, 12, 4161–4170.
27. J. C. Scaiano, M. Laferrière, R. E. Galian, V. Maurel and P. Billone, Non-linear effects in the quenching of fluorescent semiconductor nanoparticles by paramagnetic species, Phys. Status Solidi A, 2006, 203, 1337–1343.
28. J. Keizer, Nonlinear fluorescence quenching and the origin of positive curvature in Stern–Volmer plots, J. Am. Chem. Soc., 1983, 105, 1494–1498.
29. D. G. Chris, Optical halide sensing using fluorescence quenching: theory, simulations and applications – a review, Meas. Sci. Technol., 2001, 12, R53.
30. C. Gu and K. G. Karthikeyan, Interaction of tetracycline with aluminum and iron hydrous oxides, Environ. Sci. Technol., 2005, 39, 2660–2667.
31. J. R. Lacher, J. L. Bitner and J. D. Park, The infrared absorption spectra of some antibiotics in antimony trichloride solution, J. Phys. Chem., 1955, 59, 610–614.
32. H. M. Myers, H. J. Tochon-Danguy and C. A. Baud, IR absorption spectrophotometric analysis of the complex formed by tetracycline and synthetic hydroxyapatite, Calcif. Tissue Int., 1983, 35, 745–749.
33. M. K. Rai, S. D. Deshmukh, A. P. Ingle and A. K. Gade, Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria, J. Appl. Microbiol., 2012, 112, 841–852.
34. C. Marambio-Jones and E. V. Hoek, A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment, J. Nanopart. Res., 2010, 12, 1531–1551.
35. T. F. Chin and J. L. Lach, Drug diffusion and bioavailability: tetracycline metallic chelation, Am. J. Hosp. Pharm., 1975, 32, 625.

---