Materials Science and Engineering C 40 (2014) 24–31
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Release of silver and copper nanoparticles from polyethylene nanocomposites and their penetration into Listeria monocytogenes L.A. Tamayo a,⁎, P.A. Zapata a,b, N.D. Vejar a, M.I. Azócar a, M.A. Gulppi a, X. Zhou c, G.E. Thompson c, F.M. Rabagliati a,b, M.A. Páez a a b c
Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. L. B. O'Higgins 3363, Casilla 40, Correo 33, Santiago, Chile Grupo de Polímeros, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. L. B. O'Higgins 3363, Casilla 40, Correo 33, Santiago, Chile Corrosion and Protection Centre, School of Materials, The University of Manchester, Manchester, M13 9PL England, UK
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 1 March 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Silver nanoparticles Copper nanoparticles Nanocomposites Polyethylene Antibacterial properties
a b s t r a c t Since infection is a major cause of death in a patient whose immune responses have been compromised (immunocompromised patient), considerable attention has been focused on developing materials for the prevention of infections. This has been directed primarily at suppressing or eliminating the host's endogenous microbial burden and decreasing the acquisition of new organisms. In this study, the antibacterial properties of two nanocomposites, polyethylene modified with silver nanoparticles (PE-AgNps) or copper nanoparticles (PE-CuNps), against Listeria monocytogenes have been investigated. In order to elucidate the antibacterial mechanism, specifically whether this mechanism corresponds to bactericidal or bacteriolytic activities, we have determined the extent of release of metal ions (Ag+ and Cu2+) and, also, the morphology of the bacteria. The metal ion release from nanocomposites was followed by inductively coupled plasma spectrometry and the morphology of the bacteria was revealed through examination of ultramicrotomed sections of bacteria in a transmission electron microscope. The study of metal ion release from the nanocomposites shows that for both nanocomposites the amount of ions released varies with time, which initially displays a linear behavior until an asymptotic behavior is reached. Further, TEM images show that silver nanoparticles (AgNps) and copper nanoparticles (CuNps), which are released from the nanocomposites, can penetrate to the cell wall and the plasma membrane of bacteria. Resulting morphological changes involve separation of the cytoplasmic membrane from the cell wall, which is known to be an effect of plasmolysis. It was revealed that the antibacterial abilities of the two nanocomposites against L. monocytogenes are associated with both bactericidal and bacteriolytic effects. © 2014 Published by Elsevier B.V.
1. Introduction Listeria monocytogenes is a Gram positive bacteria that causes meningitis, encephalitis, bacteremia and febrile gastroenteritis [1,2]. The main route of transmission is through consumption of contaminated food; however, there are reports of nosocomial transmission associated with contamination of medical devices [3]. Therefore, prevention of infection becomes relevant together with the methods and materials to limit or inhibit the growth of microorganisms. In this sense, the use of composite materials, which have incorporated antibacterial agents in their compositions, is being progressed as an excellent alternative in controlling the growth of microorganisms and, consequently, in the prevention of disease transmission. Polymer–nanoparticle materials are present within the composites, with the nanoparticles having the role of antibacterial agents. It is in the above context that this paper is framed. Several previous investigations have focused on studying the ⁎ Corresponding author. E-mail address: [email protected] (L.A. Tamayo).
http://dx.doi.org/10.1016/j.msec.2014.03.037 0928-4931/© 2014 Published by Elsevier B.V.
potential of AgNps and CuNps for application in the development of materials with efficient and effective biocidal performance against bacteria such as Staphylococcus aureus [4], Escherichia coli [5], Klebsiella pneumonia [6], Pseudomonas aeruginosa [7], Enterobacter cloacae [8], Salmonella typhimurium [9] and L. monocytogenes [10]. The mechanism by which the nanoparticles promote this antibacterial ability, so far, is the subject of debate. At least three mechanisms were cited [11–16], including morphological and structural changes in the bacterial cells, attack of the respiratory chain and formation of regions of low molecular weight within the bacteria. However, most studies pointed to the bactericidal effect of silver ions that are released from the nanoparticles [17]. In this regard, a number of studies have focused on understanding the mechanism of release of ions from the nanoparticles, showing that control of the release of ions depends on factors such as, type of stabilizing agent and the chemical nature of the medium [18,19]. The antibacterial ability of AgNps in suspension is explained by two mechanisms. The first considers adhesion of the nanoparticles to the bacteria surface, resulting in cell wall damage and, in some cases, penetration through the cell wall, when the nanoparticles are smaller than
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
10 nm, into bacteria [16,20]. Other studies have shown that AgNps led to the production of silver ions and, subsequently, reactive oxygen species that are the main source of toxicity [21]. Concerning the antibacterial ability of CuNps in suspension, the mechanism is less known. Recent reports have suggested that the antibacterial activity is related to Cu2+ ion release, which would cause the disruption of the plasmatic membrane of bacteria, enabling their entry into the bacteria and alteration of the bacteria enzymatic functions [22,23]. The antibacterial behavior of polymeric materials modified with antibacterial agents, such as AgNps and CuNps, is even less known. The antibacterial behavior of metal–polymer nanocomposites has been attributed to the release of ions from the nanoparticles. The release is preceded by the diffusion of water into the polymer and the oxidation of nanoparticles. The released ions penetrate to the bacterial cell wall. Two mechanisms have been suggested to explain the antibacterial ability of the modified polymers. The first mechanism considers that bacteria are killed by the release of silver ions from the nanoparticles (bactericidal effect) and the second mechanism proposed that the suspended nanoparticles inhibit the survival and reproduction of bacteria (bacteriostatic effect) [24]. In order to improve the understanding of the antibacterial ability of the polymer–AgNp and polymer–CuNp systems, the present study examines systematically the antibacterial activity of these nanocomposites against L. monocytogenes (ISP 6508) bacteria. It was found that the antibacterial ability of the nanocomposites is associated with the release of the metal ions and, also, the penetration of metallic nanoparticles in the bacterial cells. Thus, for the nanocomposites, a dual antibacterial effect is proposed. 2. Experimental section 2.1. Materials All solutions were prepared with deionized water. AgNO3 (96%), CuCl2 (99.8%), oleic acid (99%), sodium citrate (98%), NaBH4 and toluene (ACS reagent grade, N99.5%) were supplied by Aldrich. KH2PO4 (99.4%), NaCl (99%) and surfactant Tween 80 were supplied by Merck. The metallocene catalyst, ethenyl(bisindenil)zirconium dichloride, combined with methylaluminoxane (MAO, 10% toluene solution) was used as a co-catalyst system for in-situ ethylene polymerization. 2.2. Preparation of silver nanoparticles AgNps were prepared by chemical reduction of silver nitrate with sodium borohydride in the presence of oleic acid, which was used as a surfactant, according to the method used by Wang et al. [25]. Silver and copper nanoparticles were analyzed in a JEOL 2010 TEM equipped with an energy dispersive X-ray spectrometer (EDS) at an accelerating voltage of 200 kV. 2.3. Preparation of copper nanoparticles CuNps were prepared by the chemical reduction of CuCl2 with sodium borohydride in the presence of sodium citrate as a surfactant according to the following procedure. 1 ml of 0.04 M CuCl2 was added to 50 ml of 0.1 M sodium citrate; 0.5 ml of 0.5 M sodium borohydride was then added to the stirred solution, changing the color from blue to coffee. The reaction was performed under a nitrogen atmosphere to prevent oxidation of the copper. 2.4. Polymerization of ethylene Polymerization of ethylene was carried out in a 400 ml glass reactor with mechanical stirring and temperature control. Toluene, MAO and the metallocene catalyst solution in toluene were added to the reactor,
25
using a syringe under the nitrogen atmosphere. The nitrogen atmosphere was then eliminated and replaced by ethylene at a pressure of 1 bar. In each experiment, 3 × 10−6 mol of metallocene catalyst and 2.6 ml (4.32 × 10− 3 mol) of MAO toluene solution, ([Al] / [Zr] = 1400) were used. The final volume of the solution in the reactor was 80 ml. The polymerization was carried out at 60 °C and 1 bar of ethylene pressure for 30 min, with stirring at 500 rpm. The polymerization was terminated by addition of acidified methanol (10% HCl, 10 ml). The polyethylene product was recovered by filtration, then washed with methanol and dried under vacuum at room temperature for 12 h.
2.5. Preparation of nanocomposites The preparation of nanocomposites was conducted using the same conditions for ethylene polymerization. Toluene, 2.6 ml of MAO and then, AgNps or CuNps, both dispersed in toluene, were added to the reactor. Subsequently, the metallocene solution was added and the reaction solution was saturated with ethylene. The nanocomposites were prepared with different amounts of AgNps and CuNps (1 to 5 wt.% relative to the weight of the virgin polyethylene).
2.6. Antimicrobial assessment 2.6.1. Bacterial viability The antibacterial ability was assessed against L. monocytogenes (ISP 6508). Bacteria were grown in the culture broth of Mueller Hinton at 37 °C for 16 h. The resultant culture was then transferred to a fresh medium and standardized to 106 CFU/ml by measuring the optical density at 600 nm. One drop of this solution was deposited on the surface of the nanocomposites, of dimensions 1 × 1 cm2, and then incubated for 6, 10 and 24 h. Samples were washed with 10 ml of a solution containing 0.88 wt.% NaCl and 1 wt.% Tween 80. This was undertaken, in order to drag the bacteria incubated in the surface of the nanocomposite. Subsequently, 40 μl of the solution with entrained bacteria was transferred to a nutrient medium, Mueller Hinton agar, and incubated for 16 h at 37 ºC. After incubation, colonies were counted. The experiments were repeated 4 times.
2.6.2. Transmission electron microscopy An aliquot of 3 ml of L. monocytogenes (ISP 6508) solution, standardized to 108 CFU/ml, was deposited on PE-AgNp or PE-CuNp nanocomposites of dimensions 5 × 5 cm2, and incubated for 16 h. After incubation, the samples were washed with cacodylate buffer to remove bacteria; this solution was then centrifuged at 3000 rpm to obtain a pellet of bacteria. The pellet was fixed by exposure to a 2.5% glutaraldehyde solution in a cacodylate buffer for 30 min, followed by dehydration of the bacteria using 50, 60, 70, 90 and 100% ethanol/cacodylate buffer solutions. The bacteria were finally embedded in epoxy resin that was cured at 60 °C for 24 h. The embedded samples were then sectioned using a Leica Ultracut microtome equipped with a diamond knife, to generate electron transparent slices of nominal thickness of 80 nm for examination in a JEOL 2000 TEM.
2.7. Ion release measurements The release of silver and copper ions from the nanocomposites was assessed by inductively coupled plasma (ICP) spectrometry. The nanocomposite samples, of dimensions 1 × 2 × 0.01 cm, were immersed in 10 ml of deionised water that was stirred occasionally. The measurements were performed after immersion for 1, 4, 8, 12, 15, 30, 45, 60, 75 and 90 days. The solutions were then centrifuged at 1800 rpm for 2 min to ensure the absence of any particulate material in the solution.
26
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
2.8. Characterization UV absorption spectra of the silver and copper nanoparticles in toluene solution were taken on a Shimadzu UV-1800 spectrophotometer at 0.5 s exposure times, in the range of 350–700 nm. A variable path bucket with quartz windows was used. The sizes and the degree of dispersion of the nanoparticles were assessed by transmission electron microscopy (TEM, JEOL 2000) and scanning electron microscopy (SEM, Hitachi S5500). For TEM examination, the AgNp and CuNp suspensions were deposited on a carbon-coated copper grid. The nanocomposites were ultramicrotomed to generate electron transparent sections of 80 nm thickness for TEM examination. 3. Results and discussion 3.1. Characterization of AgNps, CuNps and nanocomposites The transmission electron micrographs and UV–vis spectra of AgNps are displayed in Fig. 1, nanoparticles reveal spherical morphologies, with diameters between 5 and 15 nm and UV–vis spectra show the maximum absorbance peak at ca. 414 nm that confirming the presence of silver nanoparticles. CuNps in Fig. 2 show diameters between 2 and 4 nm, and the size distribution for CuNps is narrower than for AgNps. This is probably associated with the high concentration of sodium citrate used to stabilize the formation of CuNps. UV–vis spectra show the characteristic absorption band of copper nanoparticles at ca. 564 nm. The TEM images of nanocomposites with 5 wt.% of AgNps or CuNps are displayed in Fig. 3. AgNps in Fig. 3(a) are well distributed in the polymer matrix, which is possibly the result of the function of oleic acid as surfactant, which allows interaction with the hydrophobic tail of the polymer. Fig. 3(b) also shows a good dispersion of CuNps in the polymeric matrix. In this case, the structure of sodium citrate (surfactant of CuNps) shows no significant polar regions that could interact with the polymer matrix. However, the good dispersion obtained in the PECuNp nanocomposite could be associated with the large amount of surfactant used in the synthesis of CuNp (125 times the amount of CuCl2). In addition, the strong interaction between the carboxylate groups of the surfactant with copper [26] is expected to limit the particle–particle interaction. In order to evaluate the influence of the amount of nanoparticles on the antibacterial ability of the nanocomposites, the composites were modified with additions of 1, 2 and 5 wt.% of nanoparticles. SEM micrographs of the PE-AgNp and PE-CuNp nanocomposites are displayed in
Fig. 4. The formation of clusters of nanoparticles with sizes up to 120 nm is evident, indicating aggregation of the nanoparticles in the polymer matrix. However, the sizes of the clusters are 100 times smaller than those reported in other studies [27,28], thus indicating that the methodology used in the present study for preparing the nanocomposites facilitated a good dispersion of the nanoparticles in the polymer. 3.2. Release of silver and copper ions from nanocomposites It is known that the antibacterial mechanism of polymer–nanoparticle systems is based on the release of ions from nanoparticles in the presence of humidity and oxygen from air. Elemental nanoparticles release small amounts of ions that exhibit a biocidal activity against a broad spectrum of microorganism [29]. In this work, we follow the amount of ions released from modified nanocomposites by inductively coupled plasma spectrometry for 90 days. Fig. 5(a) and (b) shows the ion release curves of PE-AgNps and PE-CuNps, respectively, with 2 and 5 wt.% of nanoparticles. During the first 8 days of immersion, the releases of low and controlled amounts of ions are observed, which is probably attributable to the rate-determining step that is primarily associated with the penetration of water molecules into the nanocomposite [31]. Subsequently, between 9 and 15 days, the amounts of silver and copper ions increased, reaching a significant release rate of 3.36 and 0.012 mg l−1 cm−2 per day respectively, to achieve, at day 20, an asymptotic limit. At the termination of the experiment, i.e. after 90 days immersion, the amounts of ions released from the nanocomposites with 5 wt.% AgNps and CuNps are 0.36 and 90.9 mg l−1 cm−2 respectively. The PE-CuNp nanocomposites released 253 times more ions than the AgNp nanocomposites. Whereas the polymer matrix for the two types of nanocomposites, PE-AgNps and PE-CuNps, is the same, the difference in the ability to release ions from the nanocomposites may be related to the high susceptibility of copper to oxidation. It is interesting to compare the extent of release of silver ions from PE-AgNps observed in this study with those reported from nanocomposites wherein the polymer matrix has polar characteristics. For nanocomposites of polyamide with 2 wt.% AgNps and an immersion time of 15 days, Damm et al. [30] reported a silver ion concentration of ∼ 0.22 mg l− 1 cm− 2 that was associated with their release from the polyamide nanocomposite. This value is similar to the amount of Ag+ ions released from the nanocomposites prepared in this work after an immersion time of 15 days. It appears that the homogenous and low aggregation of the AgNps in the polymer matrix, obtained with the in-situ polymerization technique, promotes oxidation and dissolution. This
Fig. 1. (a) Transmission electron micrographs revealing the morphology of the AgNps and (b) UV–vis spectra of the AgNps.
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
27
Fig. 2. (a) Transmission electron micrographs revealing the morphology of the CuNps and (b) UV–vis spectra of the CuNps.
would be expected considering the increased surface area resulting from the reduction in size of the AgNps. 3.3. Bacterial viability Fig. 6(a) shows two important behaviors regarding the antimicrobial mechanism of PE-AgNps against L. monocytogenes strain (ISP 6508). The first is the percentage decrease in viability with increasing amount of nanoparticles in the polymer. Thus, at 6 h of incubation, the nanocomposites with 1 and 2 wt.% of AgNps enable viability of 67.3 and 39.1%, and the nanocomposite with 5 wt.% of AgNps presents a viability of less than 10%. The second behavior observed in Fig. 6(a) is the increase of the biocidal properties of the PE-AgNps with the incubation time of the bacteria. For the 5 wt.% AgNp nanocomposite, an incubation time of 6 h achieves over 90% reduction of the bacterial population, while an incubation time of 24 h shows no surviving colonies. This is consistent with the study of ion release from the nanocomposite shown in Fig. 5. Fig. 6(b) displays the bacterial viability assays performed with the PE-CuNps, showing a trend-biocide similar to that presented by the PE-AgNp nanocomposite, but with more effective biocidal capability than that observed for PE-AgNps. Thus, the bacterial viability percentages decrease with increasing CuNps in the polymer, but the incubation times and the amount of CuNps required to inhibit 99.9% of the bacterial
population are lower compared with the PE-AgNp nanocomposites. This is attributed to the significant increase of Cu2+ ions released from the PE-CuNps compared with PE-AgNps. Regarding the biocidal properties of both nanocomposites, it is important to note that although viability assays show that PE-CuNps present better antibacterial properties than the PE-AgNps, the ion release measurement demonstrated that the PE-AgNps is more efficient. For example, at 24 h of incubation and with 5 wt.% nanoparticles in the PE-AgNps and PE-CuNps, and with the same percentages of viability, the amounts of released Ag+ and Cu2 + ions were 0.085 and 17.28 mg l− 1 cm−2 respectively. This indicates that the PE-AgNps reach the same antibacterial effect with release of a lower dose of Ag+ ions, suggesting greater susceptibility of L. monocytogenes strain (ISP 6508) to the antibacterial action of Ag+ ions. Some work [4] has attributed this response to Ag+ ion affinity with the peptidoglycan layer, which is part of the cell membrane of Gram positive bacteria. Considering other studies [30,31], the effectiveness of the biocidal nanocomposite PE-AgNps studied here is significant. It is known that the hydrophilic nature of some nanocomposites, for example polyamide/AgNp system [31], facilitates the release of ions with respect to hydrophobic nanocomposites [30]. Contrary to the above statement, the amount of ions released from the PE-AgNps prepared in this work, which are clearly of hydrophobic nature, was comparable to those reported for hydrophilic nanocomposites. It is then apparent that for ion
Fig. 3. Transmission electron micrographs revealing the morphology of the nanocomposites: (a) PE-AgNp nanocomposite; (b) PE-CuNp nanocomposite.
28
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 4. Scanning electron micrographs showing the morphology of PE-AgNp and PE-CuNp nanocomposites with different amounts of nanoparticles: (a, d) 1 wt.%; (b, e) 2 wt.%; (c, f) 5 wt.%.
release the dispersion of nanoparticles in the matrix is important along with the hydrophilicity of the nanocomposite. A high dispersion is expected to increase the surface area for ion release. The confocal microscopy images (Fig. 1S, Supplementary data) show L. monocytogenes on the surface of polyethylene and PE-AgNp nanocomposites with different amounts of nanoparticles. Clearly the number of dead bacteria increases with the amount of nanoparticles in the polymer, while the number of living bacteria decreases. This result confirms the observed dose-dependent behavior on bacterial viability assays. A similar response is observed for PE-CuNp nanocomposites (Fig. 2S, Supplementary data) and, particularly, for the nanocomposite with 5 wt.% of nanoparticles. In the latter, there is a significant decrease in the population of dead bacteria compared with the nanocomposite with 2 wt.% of nanoparticles, which could indicate the likely loss of structural integrity of the bacteria. In order to clarify this phenomenon, ultramicrotomed sections of L. monocytogenes incubated on the PEAgNp and PE-CuNp surfaces were examined by TEM. Fig. 7 shows transmission electron micrographs of ultramicrotomed sections of L. monocytogenes, which were incubated on the surface of PE-AgNps (Fig. 7a and b) and PE-CuNps (Fig. 7c and d). The micrographs show two types of morphologies as follows: 1. cell wall separated from the cytoplasmic membrane, but intact, with the formation of low molecular weight regions in the center of the bacteria; 2. cell wall and membrane damage, with release of cytoplasmic material (plasmolysis effect). In bacteria with the cell wall and membrane intact, the presence of nanoparticles (dark areas), of approximately 50 nm diameter, inside the bacterium is revealed. The greater nanoparticle size compared with those observed for the nanoparticles in suspension (Fig. 1) is possibly the result of nanoparticle aggregation and clustering processes. In the case of the bacteria incubated on the PE-CuNp nanocomposite, TEM images revealed nanoparticles of 20 nm diameter within the bacteria, which is smaller than the value obtained in bacteria incubated on the
PE-AgNp nanocomposite. This is consistent with the differences in sizes observed for the nanoparticles in suspension (Fig. 2). In light of the above information, the antibacterial effect of the nanocomposites modified with 5 wt.% of nanoparticles is apparently the result of a bactericidal mechanism and a bacteriolytic antibacterial mechanism. This latter mechanism is associated with the disruption of the cell wall, the inner membrane and the cytoplasmic material of L. monocytogenes. Previous studies have investigated the biocide effect of silver ions against bacteria Gram positives such as S. aureus (ATCC 25923) [32], showing cell lysis with broken walls and membranes. The antibacterial effect of AgNps against L. monocytogenes has also been investigated [33]. In this case, the antibacterial capacity of the AgNps is associated with the strong interaction between the nanoparticles and the peptidoglycan layer of the bacterium, which contributes to the trapping of free Ag+ ions. However, until now, no evidence of penetration of nanoparticles into L. monocytogenes was available. The antibacterial effect of CuNps was also studied against Gram positive bacteria, resulting in morphological changes including deformation of the cell wall and cytoplasmic membrane [9], consistent with the observations in Fig. 7c and d for L. monocytogenes. It is probable that the penetration of the nanoparticles in the bacteria accelerates the destabilization of the cell wall and the membrane of the bacteria, possibly through the release of metal ions in its interior. Regarding the mechanism for penetration of nanoparticles, the main ways to translocate nanoparticles through membranes are two-fold: endocytosis and direct diffusion [34,35]. It has been established that nanoparticles between 10 and 100 nm, usually cross the membrane by endocytosis [36,37]. This phenomenon can be divided into three parts: first particles sticking to the membrane, second the membrane wrapping the nanoparticles, and finally the particle–lipid complex detaching from the membrane. In the case of very small nanoparticles,
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 5. Ion release from nanocomposites with different amounts of nanoparticles: (a) Ag+ release from PE-AgNps; (b) Cu2+ release from PE-CuNps.
whose diameters are only several nanometers or even smaller, endocytosis is not an effective way of penetration due to lower adhesion energy which comes mainly from the ligand–receptor interaction. Increasing the energy of bending and stretching resulting from the deformation of the membrane, cannot be compensated and in this case, the nanoparticles may aggregate to be endocytosed.
29
Theoretical studies by Li et al. argue that the hydrophilic/hydrophobic properties of nanoparticles are an important factor for its interaction with membranes, due to the lipid headgroup–nanoparticle interaction [38]. When the interaction is strong, the nanoparticle can be completely engulfed by the membrane, similar to the process of endocytosis [39]. In contrast, hydrophobic nanoparticles, such as fullerenes and nanotubes, can be inserted into the membrane, driven by its preference to the hydrophobic tail of the lipid structure. Further, considering the role of the hydrophobic/hydrophilic properties of the nanoparticles in the mechanism of penetration of them into biomembranes, Ding et al. conducted a theoretical design of a new type of nanoparticle. In the design, the authors added ligands to the nanoparticle surface, through dynamic bonds [40], which are defined as any class of bond (covalent and noncovalent) that can selectively undergo reversible breaking and reformation, usually under equilibrium conditions. Dynamic covalent bond cannot access its reversibility without a catalyst (or stimulus), while noncovalent dynamic bond is highly susceptible to thermal conditions, solvents/reagents. According to the theoretical approach, when the medium (solvent) is hydrophobic, the ligand–nanoparticle may have a hydrophobic surface. This occurs when the hydrophilic head group oriented toward the surface of the nanoparticle, while their hydrophobic tail is oriented outward, thus achieving spontaneously be inserted within the membrane. Once the nanoparticle–ligand is inside, the ligand is separated from the nanoparticle preferring oriented along the distribution of the lipid layer, then dominates the reverse reaction, according to the Le Chatelier principle, where the bare nanoparticle tends to leave the hydrophobic environment within the membrane, being expelled to the other sides, and thus completing its penetration into the membrane. Relating the theoretical approach with the results of our work, the amphiphilic ligand can be perfectly represented by the structure of oleic acid used as a stabilizer in the synthesis of silver nanoparticles. Oleic acid has a hydrophilic head group of the carboxylic acid bonded to the surface of the nanoparticle, and a hydrophobic alkyl chain tail facing the external medium. On the other hand, regarding the structure of the membrane of L. monocytogenes, its classification as Gram positive bacteria indicates the presence of an outer cell wall comprised mainly of a thick layer of peptidoglycan, of mostly hydrophobic character, and an innermost layer, plasma-lipid. Taking both factors into account, the hydrophobic characteristics of the oleic acid, and the hydrophobic properties associated with the composition of the biomembrane of the bacteria, the penetration mechanism of the nanoparticle proposed by Ding et al. [40] could be applied. Furthermore, considering the small size of copper nanoparticles (less than 5 nm), and the hydrophilic character of the sodium citrate used as a stabilizer
Fig. 6. Viability of Listeria monocytogenes ISP 6508 in the presence of (a) PE-AgNp and (b) PE-CuNp nanocomposites, incubated for 6, 12 and 24 h.
30
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 7. Listeria monocytogenes (ISP 6508) incubated for 16 h on the nanocomposite surface: (a, b) PE-AgNp nanocomposite; (c, d) PE-CuNp nanocomposite.
of the nanoparticles, it is likely that the process of penetration in this case is caused by a phenomenon of endocytosis, preceded by the aggregation of nanoparticles. 4. Conclusions The PE-AgNp and PE-CuNp nanocomposites showed excellent antibacterial activity against L. monocytogenes ISP 6508. Bacterial viability studies and confocal microscopy revealed that the antibacterial ability of the nanocomposites is dependent on the amount of nanoparticles and the incubation time. The PE-CuNp nanocomposites showed an increased antibacterial effect compared with PE-AgNps, which is attributed to the increased capacity of the PE-CuNp nanocomposite for release of ions. While most of the work attributed the antibacterial action of the nanocomposites to the release of silver ions from AgNps, it is demonstrated that the penetration of AgNps and CuNps also participates in the biocidal capability of the nanocomposite. The TEM images revealed that the antibacterial properties of PE-AgNps and PE-CuNps involve the bactericidal and bacteriolytic effects. Acknowledgments This work was supported by ANILLO (ACT 95). M.A. Páez and M. I. Azócar are also grateful to CONICYT (Grant 79090024). The authors also wish to thank the UK Engineering and Physical Sciences Research Council for provision of financial support for the LATEST2 Programme Grant and its associated characterization facilities. P. Zapata and Franco M. Rabagliati acknowledge the financial support under CONICYT insertion project 79100010. L.A. Tamayo also acknowledge to FONDECYT for postdoctoral project (Grant 3140099). Appendix A. Supplementary material Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.03.037.
References [1] D.M. Frye, R. Zweig, J. Sturgeon, M. Tormey, M. LeCavalier, I. Lee, L. Lewani, M. Laurene, An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes, Clin. Infect. Dis. 35 (2002) 943–949. [2] F. Allerberger, M. Wagner, Listeriosis: a resurgent foodborne infection, Clin. Microbiol. Infect. 16 (2010) 16–23. [3] H. Hof, R. Lampidis, J. Bensch, Nosocomial Listeria gastroenteritis in a newborn, confirmed by random amplification of polymorphic DNA, Clin. Microbiol. Infect. 6 (2000) 683–686. [4] M. Jokar, R.R. Abdul, N.A. Ibrahim, L.C. Abdullah, C.P. Tan, Melt production and antimicrobial efficiency of low-density polyethylene (LDPE)-silver nanocomposite film, Food Bioprocess. Tech. 5 (2012) 719–728. [5] P. Zapata, L. Tamayo, M. Páez, E. Cerda, I. Ázocar, F.M. Rabagliati, Nanocomposites based on polyethylene and nanosilver particles produced by metallocenic “in situ” polymerization: synthesis, characterization and antimicrobial behavior, Eur. Polym. J. 47 (2011) 1541–1549. [6] K. Vimala, Y.M. Muhan, S.K. Sivudu, K. Varaprasad, S. Ravindra, N.N. Reddy, Y. Padma, B. Sreedhar, K. MohanaRaju, Fabrication of porous chitosan films impregnated with silver nanoparticles: a facile approach for superior antibacterial application, Colloids Surf. B 76 (2010) 248–258. [7] N. Perkas, G. Amirian, S. Dubinsky, S. Gazit, A Gedanken, ultrasound-assisted coating of nylon 6,6 with silver nanoparticles and its antibacterial activity, J. Appl. Polym. Sci. 104 (2007) 1423–1430. [8] Y.H. Kim, D.K. Lee, H.G. Cha, C.W. Kim, Y.S. Kang, Synthesis and characterization of antibacterial Ag–SiO2 nanocomposite, J. Phys. Chem. C 111 (2007) 3629–3635. [9] G. Cárdenas, J. Diaz, M.F. Meléndrez, C. Cruzat, A. García, Colloidal Cu nanoparticles/ chitosan composite film obtained by microwave heating for food package applications, Polym. Bull. 62 (2009) 511–524. [10] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties, Chem. Mater. 17 (2005) 5255–5262. [11] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275 (2004) 177–182. [12] K.H. Cho, J.E. Park, T. Osaka, S.J. Park, The study of antimicrobial activity and preservative effects of nanosilver ingredient, Electrochim. Acta 51 (2005) 956–960. [13] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobial, Biotechnol. Adv. 27 (2009) 76–83. [14] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, K.P. Tam, J. Chiu, C. Che, Silver nanoparticles: partial oxidation and antibacterial activities, J. Biol. Inorg. Chem. 12 (2007) 527–534. [15] K.Y. Wong, X. Liu, Silver nanoparticles—the real “silver bullet” in clinical medicine? Med. Chem. Commun. 1 (2010) 125–131. [16] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramirez, M.J. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 (2005) 2346–2353.
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31 [17] W.R. Li, X.B. Xie, Q.S. Shi, H.Y. Zeng, Y.S. OU-Yang, Y.B. Chen, Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli, Appl. Microbiol. Biotechnol. 85 (2010) 1115–1122. [18] F. Rispoli, A. Angelov, D. Badia, A. Kumar, S. Seal, V. Shah, Understanding the toxicity of aggregated zero valent copper nanoparticles against Escherichia coli, J. Hazard. Mater. 180 (2010) 212–216. [19] J. Fabrega, S.R. Fawcett, J.C. Renshaw, J.R. Lead, Interactions of silver nanoparticles with Pseudomonas putida biofilms, Environ. Sci. Technol. 43 (2009) 9004–9009. [20] A.M. El Badawy, R.G. Silva, B. Morris, K.G. Scheckel, M.T. Suidan, T.M. Tolaymat, Surface charge-dependent toxicity of silver nanoparticles, Environ. Sci. Technol. 45 (2011) 283–287. [21] E.T. Hwang, J.H. Lee, Y.J. Chae, Y.S. Kim, B.C. Kim, B.I. Sang, M.B. Gu, Analysis of the toxic mode of action of silver nanoparticles using stress-specific bioluminescent bacteria, Small 4 (2008) 746–750. [22] Z.M. Xiu, J. Ma, P.J.J. Álvarez, Differential effect of common ligands and molecular oxygen on antimicrobial activity of silver nanoparticles versus silver ions, Environ. Sci. Technol. 45 (2011) 9003–9008. [23] C. Gunawan, W.Y. Teoh, C.P. Marquis, R. Amal, Cytotoxic origin of copper(II) oxide nanoparticles: comparative studies with micron-sized particles, leachate, and metal salts, ACS Nano 5 (2011) 7214–7225. [24] C. Damm, H. Munstedt, A. Rosch, The antimicrobial efficacy of polyamide 6/silvernano- and microcomposites, Mater. Chem. Phys. 108 (2008) 61–66. [25] W. Wang, S. Efrima, O. Regev, Directing oleate stabilized nanosized silver colloid into organic phases, Langmuir 14 (1998) 602–610. [26] W. Maketon, K.L. Ogden, Synergistic effects of citric acid and polyethyleneimine to remove copper from aqueous solutions, Chemosphere 75 (2009) 206–211. [27] E. Fages, J. Pascual, O. Fenollar, D. García-Sanoguera, R. Balart, Study of antibacterial properties of polypropylene filled with surfactant-coated silver nanoparticles, Polym. Eng. Sci. 51 (2011) 804–811. [28] S. Cai, X. Xia, C. Zhu, C. Xie, Cupric ion release controlled by copper/low density polyethylene nanocomposite in simulated uterine solution, J. Biomed. Mater. Res. Part B 80B (2007) 220–225.
31
[29] A. Panacek, L. Kvitek, R. Prucek, M. Kolar, R. Vecerova, N. Pizurova, V.K. Sharma, T. Nevecna, R. Zboril, Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity, J. Phys. Chem. B 110 (2006) 16248–16253. [30] C. Damm, H. Munstedt, A. Rosch, Long-term antimicrobial polyamide 6/silvernanocomposites, J. Mater. Sci. 42 (2007) 6067–6073. [31] C. Damm, H. Munstedt, Kinetic aspects of the silver ion release from antimicrobial polyamide/silver nanocomposites, Appl. Phys. A 91 (2008) 479–486. [32] W.K. Jung, H.C. Koo, K.W. Kim, S. Shin, S.H. Kim, Y.H. Park, Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli, Appl. Environ. Microbiol. 74 (2008) 2171–2178. [33] S. Egger, R.P. Lehmann, M.J. Height, M.J. Loessner, M. Schuppler, Antimicrobial properties of a novel silver–silica nanocomposite material, Appl. Environ. Microbiol. 75 (2009) 2973–2976. [34] P.R. Leroueil, S. Hong, A. Mecke, J.R. Baker, B.G. Orr, M.M.B. Holl, Nanoparticle interaction with biological membranes: does nanotechnology present a Janus face? Acc. Chem. Res. 40 (2007) 335–342. [35] K. Yang, Y.Q. Ma, Computer simulation of the translocation of the nanoparticles with different shapes across a lipid bilayer, Nat. Nanotechnol. 5 (2010) 579–583. [36] L. Rajendran, H.J. Knolker, K. Simons, Subcellular targeting strategies for drug design and delivery, Nat. Rev. Drug Discov. 9 (2010) 29–42. [37] H. Gao, W.D. Shi, L.B. Freund, Mechanism of receptor-mediated endocytosis, Proc. Natl. Acad. Sci. 102 (2005) 3213–3218. [38] Y. Li, X. Chen, N. Gu, Computational investigation of interaction between nanoparticles and membranes: hydrophobic/hydrophilic effect, J. Phys. Chem. B 112 (2008) 16647–16653. [39] K.A. Smith, D. Jasnow, A.C. Balazs, Designing synthetic vesicles that engulf nanoscopic particles, J. Chem. Phys. 127 (2007) 084703. [40] H.-M. Ding, W.-D. Tian, Y.-Q. Ma, Designing nanoparticle translocation through membranes by computer simulations, ACS Nano 6 (2012) 1230–1238.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Release of silver and copper nanoparticles from polyethylene nanocomposites and their penetration into Listeria monocytogenes L.A. Tamayo a,⁎, P.A. Zapata a,b, N.D. Vejar a, M.I. Azócar a, M.A. Gulppi a, X. Zhou c, G.E. Thompson c, F.M. Rabagliati a,b, M.A. Páez a a b c
Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. L. B. O'Higgins 3363, Casilla 40, Correo 33, Santiago, Chile Grupo de Polímeros, Facultad de Química y Biología, Universidad de Santiago de Chile, Av. L. B. O'Higgins 3363, Casilla 40, Correo 33, Santiago, Chile Corrosion and Protection Centre, School of Materials, The University of Manchester, Manchester, M13 9PL England, UK
a r t i c l e
i n f o
Article history: Received 23 September 2013 Received in revised form 1 March 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Silver nanoparticles Copper nanoparticles Nanocomposites Polyethylene Antibacterial properties
a b s t r a c t Since infection is a major cause of death in a patient whose immune responses have been compromised (immunocompromised patient), considerable attention has been focused on developing materials for the prevention of infections. This has been directed primarily at suppressing or eliminating the host's endogenous microbial burden and decreasing the acquisition of new organisms. In this study, the antibacterial properties of two nanocomposites, polyethylene modified with silver nanoparticles (PE-AgNps) or copper nanoparticles (PE-CuNps), against Listeria monocytogenes have been investigated. In order to elucidate the antibacterial mechanism, specifically whether this mechanism corresponds to bactericidal or bacteriolytic activities, we have determined the extent of release of metal ions (Ag+ and Cu2+) and, also, the morphology of the bacteria. The metal ion release from nanocomposites was followed by inductively coupled plasma spectrometry and the morphology of the bacteria was revealed through examination of ultramicrotomed sections of bacteria in a transmission electron microscope. The study of metal ion release from the nanocomposites shows that for both nanocomposites the amount of ions released varies with time, which initially displays a linear behavior until an asymptotic behavior is reached. Further, TEM images show that silver nanoparticles (AgNps) and copper nanoparticles (CuNps), which are released from the nanocomposites, can penetrate to the cell wall and the plasma membrane of bacteria. Resulting morphological changes involve separation of the cytoplasmic membrane from the cell wall, which is known to be an effect of plasmolysis. It was revealed that the antibacterial abilities of the two nanocomposites against L. monocytogenes are associated with both bactericidal and bacteriolytic effects. © 2014 Published by Elsevier B.V.
1. Introduction Listeria monocytogenes is a Gram positive bacteria that causes meningitis, encephalitis, bacteremia and febrile gastroenteritis [1,2]. The main route of transmission is through consumption of contaminated food; however, there are reports of nosocomial transmission associated with contamination of medical devices [3]. Therefore, prevention of infection becomes relevant together with the methods and materials to limit or inhibit the growth of microorganisms. In this sense, the use of composite materials, which have incorporated antibacterial agents in their compositions, is being progressed as an excellent alternative in controlling the growth of microorganisms and, consequently, in the prevention of disease transmission. Polymer–nanoparticle materials are present within the composites, with the nanoparticles having the role of antibacterial agents. It is in the above context that this paper is framed. Several previous investigations have focused on studying the ⁎ Corresponding author. E-mail address: [email protected] (L.A. Tamayo).
http://dx.doi.org/10.1016/j.msec.2014.03.037 0928-4931/© 2014 Published by Elsevier B.V.
potential of AgNps and CuNps for application in the development of materials with efficient and effective biocidal performance against bacteria such as Staphylococcus aureus [4], Escherichia coli [5], Klebsiella pneumonia [6], Pseudomonas aeruginosa [7], Enterobacter cloacae [8], Salmonella typhimurium [9] and L. monocytogenes [10]. The mechanism by which the nanoparticles promote this antibacterial ability, so far, is the subject of debate. At least three mechanisms were cited [11–16], including morphological and structural changes in the bacterial cells, attack of the respiratory chain and formation of regions of low molecular weight within the bacteria. However, most studies pointed to the bactericidal effect of silver ions that are released from the nanoparticles [17]. In this regard, a number of studies have focused on understanding the mechanism of release of ions from the nanoparticles, showing that control of the release of ions depends on factors such as, type of stabilizing agent and the chemical nature of the medium [18,19]. The antibacterial ability of AgNps in suspension is explained by two mechanisms. The first considers adhesion of the nanoparticles to the bacteria surface, resulting in cell wall damage and, in some cases, penetration through the cell wall, when the nanoparticles are smaller than
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
10 nm, into bacteria [16,20]. Other studies have shown that AgNps led to the production of silver ions and, subsequently, reactive oxygen species that are the main source of toxicity [21]. Concerning the antibacterial ability of CuNps in suspension, the mechanism is less known. Recent reports have suggested that the antibacterial activity is related to Cu2+ ion release, which would cause the disruption of the plasmatic membrane of bacteria, enabling their entry into the bacteria and alteration of the bacteria enzymatic functions [22,23]. The antibacterial behavior of polymeric materials modified with antibacterial agents, such as AgNps and CuNps, is even less known. The antibacterial behavior of metal–polymer nanocomposites has been attributed to the release of ions from the nanoparticles. The release is preceded by the diffusion of water into the polymer and the oxidation of nanoparticles. The released ions penetrate to the bacterial cell wall. Two mechanisms have been suggested to explain the antibacterial ability of the modified polymers. The first mechanism considers that bacteria are killed by the release of silver ions from the nanoparticles (bactericidal effect) and the second mechanism proposed that the suspended nanoparticles inhibit the survival and reproduction of bacteria (bacteriostatic effect) [24]. In order to improve the understanding of the antibacterial ability of the polymer–AgNp and polymer–CuNp systems, the present study examines systematically the antibacterial activity of these nanocomposites against L. monocytogenes (ISP 6508) bacteria. It was found that the antibacterial ability of the nanocomposites is associated with the release of the metal ions and, also, the penetration of metallic nanoparticles in the bacterial cells. Thus, for the nanocomposites, a dual antibacterial effect is proposed. 2. Experimental section 2.1. Materials All solutions were prepared with deionized water. AgNO3 (96%), CuCl2 (99.8%), oleic acid (99%), sodium citrate (98%), NaBH4 and toluene (ACS reagent grade, N99.5%) were supplied by Aldrich. KH2PO4 (99.4%), NaCl (99%) and surfactant Tween 80 were supplied by Merck. The metallocene catalyst, ethenyl(bisindenil)zirconium dichloride, combined with methylaluminoxane (MAO, 10% toluene solution) was used as a co-catalyst system for in-situ ethylene polymerization. 2.2. Preparation of silver nanoparticles AgNps were prepared by chemical reduction of silver nitrate with sodium borohydride in the presence of oleic acid, which was used as a surfactant, according to the method used by Wang et al. [25]. Silver and copper nanoparticles were analyzed in a JEOL 2010 TEM equipped with an energy dispersive X-ray spectrometer (EDS) at an accelerating voltage of 200 kV. 2.3. Preparation of copper nanoparticles CuNps were prepared by the chemical reduction of CuCl2 with sodium borohydride in the presence of sodium citrate as a surfactant according to the following procedure. 1 ml of 0.04 M CuCl2 was added to 50 ml of 0.1 M sodium citrate; 0.5 ml of 0.5 M sodium borohydride was then added to the stirred solution, changing the color from blue to coffee. The reaction was performed under a nitrogen atmosphere to prevent oxidation of the copper. 2.4. Polymerization of ethylene Polymerization of ethylene was carried out in a 400 ml glass reactor with mechanical stirring and temperature control. Toluene, MAO and the metallocene catalyst solution in toluene were added to the reactor,
25
using a syringe under the nitrogen atmosphere. The nitrogen atmosphere was then eliminated and replaced by ethylene at a pressure of 1 bar. In each experiment, 3 × 10−6 mol of metallocene catalyst and 2.6 ml (4.32 × 10− 3 mol) of MAO toluene solution, ([Al] / [Zr] = 1400) were used. The final volume of the solution in the reactor was 80 ml. The polymerization was carried out at 60 °C and 1 bar of ethylene pressure for 30 min, with stirring at 500 rpm. The polymerization was terminated by addition of acidified methanol (10% HCl, 10 ml). The polyethylene product was recovered by filtration, then washed with methanol and dried under vacuum at room temperature for 12 h.
2.5. Preparation of nanocomposites The preparation of nanocomposites was conducted using the same conditions for ethylene polymerization. Toluene, 2.6 ml of MAO and then, AgNps or CuNps, both dispersed in toluene, were added to the reactor. Subsequently, the metallocene solution was added and the reaction solution was saturated with ethylene. The nanocomposites were prepared with different amounts of AgNps and CuNps (1 to 5 wt.% relative to the weight of the virgin polyethylene).
2.6. Antimicrobial assessment 2.6.1. Bacterial viability The antibacterial ability was assessed against L. monocytogenes (ISP 6508). Bacteria were grown in the culture broth of Mueller Hinton at 37 °C for 16 h. The resultant culture was then transferred to a fresh medium and standardized to 106 CFU/ml by measuring the optical density at 600 nm. One drop of this solution was deposited on the surface of the nanocomposites, of dimensions 1 × 1 cm2, and then incubated for 6, 10 and 24 h. Samples were washed with 10 ml of a solution containing 0.88 wt.% NaCl and 1 wt.% Tween 80. This was undertaken, in order to drag the bacteria incubated in the surface of the nanocomposite. Subsequently, 40 μl of the solution with entrained bacteria was transferred to a nutrient medium, Mueller Hinton agar, and incubated for 16 h at 37 ºC. After incubation, colonies were counted. The experiments were repeated 4 times.
2.6.2. Transmission electron microscopy An aliquot of 3 ml of L. monocytogenes (ISP 6508) solution, standardized to 108 CFU/ml, was deposited on PE-AgNp or PE-CuNp nanocomposites of dimensions 5 × 5 cm2, and incubated for 16 h. After incubation, the samples were washed with cacodylate buffer to remove bacteria; this solution was then centrifuged at 3000 rpm to obtain a pellet of bacteria. The pellet was fixed by exposure to a 2.5% glutaraldehyde solution in a cacodylate buffer for 30 min, followed by dehydration of the bacteria using 50, 60, 70, 90 and 100% ethanol/cacodylate buffer solutions. The bacteria were finally embedded in epoxy resin that was cured at 60 °C for 24 h. The embedded samples were then sectioned using a Leica Ultracut microtome equipped with a diamond knife, to generate electron transparent slices of nominal thickness of 80 nm for examination in a JEOL 2000 TEM.
2.7. Ion release measurements The release of silver and copper ions from the nanocomposites was assessed by inductively coupled plasma (ICP) spectrometry. The nanocomposite samples, of dimensions 1 × 2 × 0.01 cm, were immersed in 10 ml of deionised water that was stirred occasionally. The measurements were performed after immersion for 1, 4, 8, 12, 15, 30, 45, 60, 75 and 90 days. The solutions were then centrifuged at 1800 rpm for 2 min to ensure the absence of any particulate material in the solution.
26
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
2.8. Characterization UV absorption spectra of the silver and copper nanoparticles in toluene solution were taken on a Shimadzu UV-1800 spectrophotometer at 0.5 s exposure times, in the range of 350–700 nm. A variable path bucket with quartz windows was used. The sizes and the degree of dispersion of the nanoparticles were assessed by transmission electron microscopy (TEM, JEOL 2000) and scanning electron microscopy (SEM, Hitachi S5500). For TEM examination, the AgNp and CuNp suspensions were deposited on a carbon-coated copper grid. The nanocomposites were ultramicrotomed to generate electron transparent sections of 80 nm thickness for TEM examination. 3. Results and discussion 3.1. Characterization of AgNps, CuNps and nanocomposites The transmission electron micrographs and UV–vis spectra of AgNps are displayed in Fig. 1, nanoparticles reveal spherical morphologies, with diameters between 5 and 15 nm and UV–vis spectra show the maximum absorbance peak at ca. 414 nm that confirming the presence of silver nanoparticles. CuNps in Fig. 2 show diameters between 2 and 4 nm, and the size distribution for CuNps is narrower than for AgNps. This is probably associated with the high concentration of sodium citrate used to stabilize the formation of CuNps. UV–vis spectra show the characteristic absorption band of copper nanoparticles at ca. 564 nm. The TEM images of nanocomposites with 5 wt.% of AgNps or CuNps are displayed in Fig. 3. AgNps in Fig. 3(a) are well distributed in the polymer matrix, which is possibly the result of the function of oleic acid as surfactant, which allows interaction with the hydrophobic tail of the polymer. Fig. 3(b) also shows a good dispersion of CuNps in the polymeric matrix. In this case, the structure of sodium citrate (surfactant of CuNps) shows no significant polar regions that could interact with the polymer matrix. However, the good dispersion obtained in the PECuNp nanocomposite could be associated with the large amount of surfactant used in the synthesis of CuNp (125 times the amount of CuCl2). In addition, the strong interaction between the carboxylate groups of the surfactant with copper [26] is expected to limit the particle–particle interaction. In order to evaluate the influence of the amount of nanoparticles on the antibacterial ability of the nanocomposites, the composites were modified with additions of 1, 2 and 5 wt.% of nanoparticles. SEM micrographs of the PE-AgNp and PE-CuNp nanocomposites are displayed in
Fig. 4. The formation of clusters of nanoparticles with sizes up to 120 nm is evident, indicating aggregation of the nanoparticles in the polymer matrix. However, the sizes of the clusters are 100 times smaller than those reported in other studies [27,28], thus indicating that the methodology used in the present study for preparing the nanocomposites facilitated a good dispersion of the nanoparticles in the polymer. 3.2. Release of silver and copper ions from nanocomposites It is known that the antibacterial mechanism of polymer–nanoparticle systems is based on the release of ions from nanoparticles in the presence of humidity and oxygen from air. Elemental nanoparticles release small amounts of ions that exhibit a biocidal activity against a broad spectrum of microorganism [29]. In this work, we follow the amount of ions released from modified nanocomposites by inductively coupled plasma spectrometry for 90 days. Fig. 5(a) and (b) shows the ion release curves of PE-AgNps and PE-CuNps, respectively, with 2 and 5 wt.% of nanoparticles. During the first 8 days of immersion, the releases of low and controlled amounts of ions are observed, which is probably attributable to the rate-determining step that is primarily associated with the penetration of water molecules into the nanocomposite [31]. Subsequently, between 9 and 15 days, the amounts of silver and copper ions increased, reaching a significant release rate of 3.36 and 0.012 mg l−1 cm−2 per day respectively, to achieve, at day 20, an asymptotic limit. At the termination of the experiment, i.e. after 90 days immersion, the amounts of ions released from the nanocomposites with 5 wt.% AgNps and CuNps are 0.36 and 90.9 mg l−1 cm−2 respectively. The PE-CuNp nanocomposites released 253 times more ions than the AgNp nanocomposites. Whereas the polymer matrix for the two types of nanocomposites, PE-AgNps and PE-CuNps, is the same, the difference in the ability to release ions from the nanocomposites may be related to the high susceptibility of copper to oxidation. It is interesting to compare the extent of release of silver ions from PE-AgNps observed in this study with those reported from nanocomposites wherein the polymer matrix has polar characteristics. For nanocomposites of polyamide with 2 wt.% AgNps and an immersion time of 15 days, Damm et al. [30] reported a silver ion concentration of ∼ 0.22 mg l− 1 cm− 2 that was associated with their release from the polyamide nanocomposite. This value is similar to the amount of Ag+ ions released from the nanocomposites prepared in this work after an immersion time of 15 days. It appears that the homogenous and low aggregation of the AgNps in the polymer matrix, obtained with the in-situ polymerization technique, promotes oxidation and dissolution. This
Fig. 1. (a) Transmission electron micrographs revealing the morphology of the AgNps and (b) UV–vis spectra of the AgNps.
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
27
Fig. 2. (a) Transmission electron micrographs revealing the morphology of the CuNps and (b) UV–vis spectra of the CuNps.
would be expected considering the increased surface area resulting from the reduction in size of the AgNps. 3.3. Bacterial viability Fig. 6(a) shows two important behaviors regarding the antimicrobial mechanism of PE-AgNps against L. monocytogenes strain (ISP 6508). The first is the percentage decrease in viability with increasing amount of nanoparticles in the polymer. Thus, at 6 h of incubation, the nanocomposites with 1 and 2 wt.% of AgNps enable viability of 67.3 and 39.1%, and the nanocomposite with 5 wt.% of AgNps presents a viability of less than 10%. The second behavior observed in Fig. 6(a) is the increase of the biocidal properties of the PE-AgNps with the incubation time of the bacteria. For the 5 wt.% AgNp nanocomposite, an incubation time of 6 h achieves over 90% reduction of the bacterial population, while an incubation time of 24 h shows no surviving colonies. This is consistent with the study of ion release from the nanocomposite shown in Fig. 5. Fig. 6(b) displays the bacterial viability assays performed with the PE-CuNps, showing a trend-biocide similar to that presented by the PE-AgNp nanocomposite, but with more effective biocidal capability than that observed for PE-AgNps. Thus, the bacterial viability percentages decrease with increasing CuNps in the polymer, but the incubation times and the amount of CuNps required to inhibit 99.9% of the bacterial
population are lower compared with the PE-AgNp nanocomposites. This is attributed to the significant increase of Cu2+ ions released from the PE-CuNps compared with PE-AgNps. Regarding the biocidal properties of both nanocomposites, it is important to note that although viability assays show that PE-CuNps present better antibacterial properties than the PE-AgNps, the ion release measurement demonstrated that the PE-AgNps is more efficient. For example, at 24 h of incubation and with 5 wt.% nanoparticles in the PE-AgNps and PE-CuNps, and with the same percentages of viability, the amounts of released Ag+ and Cu2 + ions were 0.085 and 17.28 mg l− 1 cm−2 respectively. This indicates that the PE-AgNps reach the same antibacterial effect with release of a lower dose of Ag+ ions, suggesting greater susceptibility of L. monocytogenes strain (ISP 6508) to the antibacterial action of Ag+ ions. Some work [4] has attributed this response to Ag+ ion affinity with the peptidoglycan layer, which is part of the cell membrane of Gram positive bacteria. Considering other studies [30,31], the effectiveness of the biocidal nanocomposite PE-AgNps studied here is significant. It is known that the hydrophilic nature of some nanocomposites, for example polyamide/AgNp system [31], facilitates the release of ions with respect to hydrophobic nanocomposites [30]. Contrary to the above statement, the amount of ions released from the PE-AgNps prepared in this work, which are clearly of hydrophobic nature, was comparable to those reported for hydrophilic nanocomposites. It is then apparent that for ion
Fig. 3. Transmission electron micrographs revealing the morphology of the nanocomposites: (a) PE-AgNp nanocomposite; (b) PE-CuNp nanocomposite.
28
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 4. Scanning electron micrographs showing the morphology of PE-AgNp and PE-CuNp nanocomposites with different amounts of nanoparticles: (a, d) 1 wt.%; (b, e) 2 wt.%; (c, f) 5 wt.%.
release the dispersion of nanoparticles in the matrix is important along with the hydrophilicity of the nanocomposite. A high dispersion is expected to increase the surface area for ion release. The confocal microscopy images (Fig. 1S, Supplementary data) show L. monocytogenes on the surface of polyethylene and PE-AgNp nanocomposites with different amounts of nanoparticles. Clearly the number of dead bacteria increases with the amount of nanoparticles in the polymer, while the number of living bacteria decreases. This result confirms the observed dose-dependent behavior on bacterial viability assays. A similar response is observed for PE-CuNp nanocomposites (Fig. 2S, Supplementary data) and, particularly, for the nanocomposite with 5 wt.% of nanoparticles. In the latter, there is a significant decrease in the population of dead bacteria compared with the nanocomposite with 2 wt.% of nanoparticles, which could indicate the likely loss of structural integrity of the bacteria. In order to clarify this phenomenon, ultramicrotomed sections of L. monocytogenes incubated on the PEAgNp and PE-CuNp surfaces were examined by TEM. Fig. 7 shows transmission electron micrographs of ultramicrotomed sections of L. monocytogenes, which were incubated on the surface of PE-AgNps (Fig. 7a and b) and PE-CuNps (Fig. 7c and d). The micrographs show two types of morphologies as follows: 1. cell wall separated from the cytoplasmic membrane, but intact, with the formation of low molecular weight regions in the center of the bacteria; 2. cell wall and membrane damage, with release of cytoplasmic material (plasmolysis effect). In bacteria with the cell wall and membrane intact, the presence of nanoparticles (dark areas), of approximately 50 nm diameter, inside the bacterium is revealed. The greater nanoparticle size compared with those observed for the nanoparticles in suspension (Fig. 1) is possibly the result of nanoparticle aggregation and clustering processes. In the case of the bacteria incubated on the PE-CuNp nanocomposite, TEM images revealed nanoparticles of 20 nm diameter within the bacteria, which is smaller than the value obtained in bacteria incubated on the
PE-AgNp nanocomposite. This is consistent with the differences in sizes observed for the nanoparticles in suspension (Fig. 2). In light of the above information, the antibacterial effect of the nanocomposites modified with 5 wt.% of nanoparticles is apparently the result of a bactericidal mechanism and a bacteriolytic antibacterial mechanism. This latter mechanism is associated with the disruption of the cell wall, the inner membrane and the cytoplasmic material of L. monocytogenes. Previous studies have investigated the biocide effect of silver ions against bacteria Gram positives such as S. aureus (ATCC 25923) [32], showing cell lysis with broken walls and membranes. The antibacterial effect of AgNps against L. monocytogenes has also been investigated [33]. In this case, the antibacterial capacity of the AgNps is associated with the strong interaction between the nanoparticles and the peptidoglycan layer of the bacterium, which contributes to the trapping of free Ag+ ions. However, until now, no evidence of penetration of nanoparticles into L. monocytogenes was available. The antibacterial effect of CuNps was also studied against Gram positive bacteria, resulting in morphological changes including deformation of the cell wall and cytoplasmic membrane [9], consistent with the observations in Fig. 7c and d for L. monocytogenes. It is probable that the penetration of the nanoparticles in the bacteria accelerates the destabilization of the cell wall and the membrane of the bacteria, possibly through the release of metal ions in its interior. Regarding the mechanism for penetration of nanoparticles, the main ways to translocate nanoparticles through membranes are two-fold: endocytosis and direct diffusion [34,35]. It has been established that nanoparticles between 10 and 100 nm, usually cross the membrane by endocytosis [36,37]. This phenomenon can be divided into three parts: first particles sticking to the membrane, second the membrane wrapping the nanoparticles, and finally the particle–lipid complex detaching from the membrane. In the case of very small nanoparticles,
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 5. Ion release from nanocomposites with different amounts of nanoparticles: (a) Ag+ release from PE-AgNps; (b) Cu2+ release from PE-CuNps.
whose diameters are only several nanometers or even smaller, endocytosis is not an effective way of penetration due to lower adhesion energy which comes mainly from the ligand–receptor interaction. Increasing the energy of bending and stretching resulting from the deformation of the membrane, cannot be compensated and in this case, the nanoparticles may aggregate to be endocytosed.
29
Theoretical studies by Li et al. argue that the hydrophilic/hydrophobic properties of nanoparticles are an important factor for its interaction with membranes, due to the lipid headgroup–nanoparticle interaction [38]. When the interaction is strong, the nanoparticle can be completely engulfed by the membrane, similar to the process of endocytosis [39]. In contrast, hydrophobic nanoparticles, such as fullerenes and nanotubes, can be inserted into the membrane, driven by its preference to the hydrophobic tail of the lipid structure. Further, considering the role of the hydrophobic/hydrophilic properties of the nanoparticles in the mechanism of penetration of them into biomembranes, Ding et al. conducted a theoretical design of a new type of nanoparticle. In the design, the authors added ligands to the nanoparticle surface, through dynamic bonds [40], which are defined as any class of bond (covalent and noncovalent) that can selectively undergo reversible breaking and reformation, usually under equilibrium conditions. Dynamic covalent bond cannot access its reversibility without a catalyst (or stimulus), while noncovalent dynamic bond is highly susceptible to thermal conditions, solvents/reagents. According to the theoretical approach, when the medium (solvent) is hydrophobic, the ligand–nanoparticle may have a hydrophobic surface. This occurs when the hydrophilic head group oriented toward the surface of the nanoparticle, while their hydrophobic tail is oriented outward, thus achieving spontaneously be inserted within the membrane. Once the nanoparticle–ligand is inside, the ligand is separated from the nanoparticle preferring oriented along the distribution of the lipid layer, then dominates the reverse reaction, according to the Le Chatelier principle, where the bare nanoparticle tends to leave the hydrophobic environment within the membrane, being expelled to the other sides, and thus completing its penetration into the membrane. Relating the theoretical approach with the results of our work, the amphiphilic ligand can be perfectly represented by the structure of oleic acid used as a stabilizer in the synthesis of silver nanoparticles. Oleic acid has a hydrophilic head group of the carboxylic acid bonded to the surface of the nanoparticle, and a hydrophobic alkyl chain tail facing the external medium. On the other hand, regarding the structure of the membrane of L. monocytogenes, its classification as Gram positive bacteria indicates the presence of an outer cell wall comprised mainly of a thick layer of peptidoglycan, of mostly hydrophobic character, and an innermost layer, plasma-lipid. Taking both factors into account, the hydrophobic characteristics of the oleic acid, and the hydrophobic properties associated with the composition of the biomembrane of the bacteria, the penetration mechanism of the nanoparticle proposed by Ding et al. [40] could be applied. Furthermore, considering the small size of copper nanoparticles (less than 5 nm), and the hydrophilic character of the sodium citrate used as a stabilizer
Fig. 6. Viability of Listeria monocytogenes ISP 6508 in the presence of (a) PE-AgNp and (b) PE-CuNp nanocomposites, incubated for 6, 12 and 24 h.
30
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31
Fig. 7. Listeria monocytogenes (ISP 6508) incubated for 16 h on the nanocomposite surface: (a, b) PE-AgNp nanocomposite; (c, d) PE-CuNp nanocomposite.
of the nanoparticles, it is likely that the process of penetration in this case is caused by a phenomenon of endocytosis, preceded by the aggregation of nanoparticles. 4. Conclusions The PE-AgNp and PE-CuNp nanocomposites showed excellent antibacterial activity against L. monocytogenes ISP 6508. Bacterial viability studies and confocal microscopy revealed that the antibacterial ability of the nanocomposites is dependent on the amount of nanoparticles and the incubation time. The PE-CuNp nanocomposites showed an increased antibacterial effect compared with PE-AgNps, which is attributed to the increased capacity of the PE-CuNp nanocomposite for release of ions. While most of the work attributed the antibacterial action of the nanocomposites to the release of silver ions from AgNps, it is demonstrated that the penetration of AgNps and CuNps also participates in the biocidal capability of the nanocomposite. The TEM images revealed that the antibacterial properties of PE-AgNps and PE-CuNps involve the bactericidal and bacteriolytic effects. Acknowledgments This work was supported by ANILLO (ACT 95). M.A. Páez and M. I. Azócar are also grateful to CONICYT (Grant 79090024). The authors also wish to thank the UK Engineering and Physical Sciences Research Council for provision of financial support for the LATEST2 Programme Grant and its associated characterization facilities. P. Zapata and Franco M. Rabagliati acknowledge the financial support under CONICYT insertion project 79100010. L.A. Tamayo also acknowledge to FONDECYT for postdoctoral project (Grant 3140099). Appendix A. Supplementary material Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.03.037.
References [1] D.M. Frye, R. Zweig, J. Sturgeon, M. Tormey, M. LeCavalier, I. Lee, L. Lewani, M. Laurene, An outbreak of febrile gastroenteritis associated with delicatessen meat contaminated with Listeria monocytogenes, Clin. Infect. Dis. 35 (2002) 943–949. [2] F. Allerberger, M. Wagner, Listeriosis: a resurgent foodborne infection, Clin. Microbiol. Infect. 16 (2010) 16–23. [3] H. Hof, R. Lampidis, J. Bensch, Nosocomial Listeria gastroenteritis in a newborn, confirmed by random amplification of polymorphic DNA, Clin. Microbiol. Infect. 6 (2000) 683–686. [4] M. Jokar, R.R. Abdul, N.A. Ibrahim, L.C. Abdullah, C.P. Tan, Melt production and antimicrobial efficiency of low-density polyethylene (LDPE)-silver nanocomposite film, Food Bioprocess. Tech. 5 (2012) 719–728. [5] P. Zapata, L. Tamayo, M. Páez, E. Cerda, I. Ázocar, F.M. Rabagliati, Nanocomposites based on polyethylene and nanosilver particles produced by metallocenic “in situ” polymerization: synthesis, characterization and antimicrobial behavior, Eur. Polym. J. 47 (2011) 1541–1549. [6] K. Vimala, Y.M. Muhan, S.K. Sivudu, K. Varaprasad, S. Ravindra, N.N. Reddy, Y. Padma, B. Sreedhar, K. MohanaRaju, Fabrication of porous chitosan films impregnated with silver nanoparticles: a facile approach for superior antibacterial application, Colloids Surf. B 76 (2010) 248–258. [7] N. Perkas, G. Amirian, S. Dubinsky, S. Gazit, A Gedanken, ultrasound-assisted coating of nylon 6,6 with silver nanoparticles and its antibacterial activity, J. Appl. Polym. Sci. 104 (2007) 1423–1430. [8] Y.H. Kim, D.K. Lee, H.G. Cha, C.W. Kim, Y.S. Kang, Synthesis and characterization of antibacterial Ag–SiO2 nanocomposite, J. Phys. Chem. C 111 (2007) 3629–3635. [9] G. Cárdenas, J. Diaz, M.F. Meléndrez, C. Cruzat, A. García, Colloidal Cu nanoparticles/ chitosan composite film obtained by microwave heating for food package applications, Polym. Bull. 62 (2009) 511–524. [10] N. Cioffi, L. Torsi, N. Ditaranto, G. Tantillo, L. Ghibelli, L. Sabbatini, T. Bleve-Zacheo, M. D'Alessio, P.G. Zambonin, E. Traversa, Copper nanoparticle/polymer composites with antifungal and bacteriostatic properties, Chem. Mater. 17 (2005) 5255–5262. [11] I. Sondi, B. Salopek-Sondi, Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria, J. Colloid Interface Sci. 275 (2004) 177–182. [12] K.H. Cho, J.E. Park, T. Osaka, S.J. Park, The study of antimicrobial activity and preservative effects of nanosilver ingredient, Electrochim. Acta 51 (2005) 956–960. [13] M. Rai, A. Yadav, A. Gade, Silver nanoparticles as a new generation of antimicrobial, Biotechnol. Adv. 27 (2009) 76–83. [14] C.N. Lok, C.M. Ho, R. Chen, Q.Y. He, W.Y. Yu, H. Sun, K.P. Tam, J. Chiu, C. Che, Silver nanoparticles: partial oxidation and antibacterial activities, J. Biol. Inorg. Chem. 12 (2007) 527–534. [15] K.Y. Wong, X. Liu, Silver nanoparticles—the real “silver bullet” in clinical medicine? Med. Chem. Commun. 1 (2010) 125–131. [16] J.R. Morones, J.L. Elechiguerra, A. Camacho, K. Holt, J.B. Kouri, J.T. Ramirez, M.J. Yacaman, The bactericidal effect of silver nanoparticles, Nanotechnology 16 (2005) 2346–2353.
L.A. Tamayo et al. / Materials Science and Engineering C 40 (2014) 24–31 [17] W.R. Li, X.B. Xie, Q.S. Shi, H.Y. Zeng, Y.S. OU-Yang, Y.B. Chen, Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli, Appl. Microbiol. Biotechnol. 85 (2010) 1115–1122. [18] F. Rispoli, A. Angelov, D. Badia, A. Kumar, S. Seal, V. Shah, Understanding the toxicity of aggregated zero valent copper nanoparticles against Escherichia coli, J. Hazard. Mater. 180 (2010) 212–216. [19] J. Fabrega, S.R. Fawcett, J.C. Renshaw, J.R. Lead, Interactions of silver nanoparticles with Pseudomonas putida biofilms, Environ. Sci. Technol. 43 (2009) 9004–9009. [20] A.M. El Badawy, R.G. Silva, B. Morris, K.G. Scheckel, M.T. Suidan, T.M. Tolaymat, Surface charge-dependent toxicity of silver nanoparticles, Environ. Sci. Technol. 45 (2011) 283–287. [21] E.T. Hwang, J.H. Lee, Y.J. Chae, Y.S. Kim, B.C. Kim, B.I. Sang, M.B. Gu, Analysis of the toxic mode of action of silver nanoparticles using stress-specific bioluminescent bacteria, Small 4 (2008) 746–750. [22] Z.M. Xiu, J. Ma, P.J.J. Álvarez, Differential effect of common ligands and molecular oxygen on antimicrobial activity of silver nanoparticles versus silver ions, Environ. Sci. Technol. 45 (2011) 9003–9008. [23] C. Gunawan, W.Y. Teoh, C.P. Marquis, R. Amal, Cytotoxic origin of copper(II) oxide nanoparticles: comparative studies with micron-sized particles, leachate, and metal salts, ACS Nano 5 (2011) 7214–7225. [24] C. Damm, H. Munstedt, A. Rosch, The antimicrobial efficacy of polyamide 6/silvernano- and microcomposites, Mater. Chem. Phys. 108 (2008) 61–66. [25] W. Wang, S. Efrima, O. Regev, Directing oleate stabilized nanosized silver colloid into organic phases, Langmuir 14 (1998) 602–610. [26] W. Maketon, K.L. Ogden, Synergistic effects of citric acid and polyethyleneimine to remove copper from aqueous solutions, Chemosphere 75 (2009) 206–211. [27] E. Fages, J. Pascual, O. Fenollar, D. García-Sanoguera, R. Balart, Study of antibacterial properties of polypropylene filled with surfactant-coated silver nanoparticles, Polym. Eng. Sci. 51 (2011) 804–811. [28] S. Cai, X. Xia, C. Zhu, C. Xie, Cupric ion release controlled by copper/low density polyethylene nanocomposite in simulated uterine solution, J. Biomed. Mater. Res. Part B 80B (2007) 220–225.
31
[29] A. Panacek, L. Kvitek, R. Prucek, M. Kolar, R. Vecerova, N. Pizurova, V.K. Sharma, T. Nevecna, R. Zboril, Silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity, J. Phys. Chem. B 110 (2006) 16248–16253. [30] C. Damm, H. Munstedt, A. Rosch, Long-term antimicrobial polyamide 6/silvernanocomposites, J. Mater. Sci. 42 (2007) 6067–6073. [31] C. Damm, H. Munstedt, Kinetic aspects of the silver ion release from antimicrobial polyamide/silver nanocomposites, Appl. Phys. A 91 (2008) 479–486. [32] W.K. Jung, H.C. Koo, K.W. Kim, S. Shin, S.H. Kim, Y.H. Park, Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli, Appl. Environ. Microbiol. 74 (2008) 2171–2178. [33] S. Egger, R.P. Lehmann, M.J. Height, M.J. Loessner, M. Schuppler, Antimicrobial properties of a novel silver–silica nanocomposite material, Appl. Environ. Microbiol. 75 (2009) 2973–2976. [34] P.R. Leroueil, S. Hong, A. Mecke, J.R. Baker, B.G. Orr, M.M.B. Holl, Nanoparticle interaction with biological membranes: does nanotechnology present a Janus face? Acc. Chem. Res. 40 (2007) 335–342. [35] K. Yang, Y.Q. Ma, Computer simulation of the translocation of the nanoparticles with different shapes across a lipid bilayer, Nat. Nanotechnol. 5 (2010) 579–583. [36] L. Rajendran, H.J. Knolker, K. Simons, Subcellular targeting strategies for drug design and delivery, Nat. Rev. Drug Discov. 9 (2010) 29–42. [37] H. Gao, W.D. Shi, L.B. Freund, Mechanism of receptor-mediated endocytosis, Proc. Natl. Acad. Sci. 102 (2005) 3213–3218. [38] Y. Li, X. Chen, N. Gu, Computational investigation of interaction between nanoparticles and membranes: hydrophobic/hydrophilic effect, J. Phys. Chem. B 112 (2008) 16647–16653. [39] K.A. Smith, D. Jasnow, A.C. Balazs, Designing synthetic vesicles that engulf nanoscopic particles, J. Chem. Phys. 127 (2007) 084703. [40] H.-M. Ding, W.-D. Tian, Y.-Q. Ma, Designing nanoparticle translocation through membranes by computer simulations, ACS Nano 6 (2012) 1230–1238.
