Au @ Ag Core @ Shell Nanopartículas sintetizadas con Rumex hymenosepalus como agente antimicrobiano

Resultados y discusiones

Caracterización

La Figura 1a muestra los espectros de absorción UV-vis de los nanomateriales sintetizados. Las absorbancias se han normalizado para la máxima resonancia de plasmón de superficie localizada (LSPR) correspondiente a cada sistema de nanopartículas.

Espectros UV-Vis de nanopartículas ( a ), Potencial Z ( b ), DLS ( c ) y PDI ( d ) de Au, Ag y Au @ AgNPs

El espectro de absorción de Au @ AgNps en la Fig. 1a tiene una sola banda centrada en 474 nm, que se encuentra entre el LSPR de AgNP (445 nm) y el LSPR de AuNP (544 nm). La ausencia de un pico de absorción similar al oro en Au @ AgNPs sugiere que los nanomateriales obtenidos por síntesis secuencial son estructuras core @ shell. No es posible detectar ninguna banda de absorción asociada con el Au pertenece al núcleo. Algunos autores asumen que para los sistemas núcleo @ caparazón, los espectros de absorción están compuestos por dos bandas asociadas con cada uno de los metales para espesores de capa entre 3 y 4 nm. La absorción asociada con el núcleo metálico desaparece para espesores más altos, obteniendo una única banda de absorción donde la ubicación máxima depende de la relación espesor / tamaño del núcleo de la partícula bimetálica [79, 80].

Samal y col. [81] sintetizó nanopartículas núcleo @ caparazón (Au @ Ag) controlando los tamaños de los núcleos y agregando capas de diferentes espesores. En particular, nuestro resultado UV-vis para Au @ AgNPs coincide con el informado por Samal et al. para núcleos de oro de 32 nm y un espesor de plata superior a 15 nm, donde los espectros se caracterizan por una sola banda de absorción (~ 450 nm), y se observa la supresión de plasmones de superficie de Au.

Además, en la Fig. 1a, se puede observar la absorción centrada en 280 nm (región resaltada en azul), correspondiente a moléculas del Rumex hymenosepalus extracto utilizado como agente reductor en nuestra síntesis de nanopartículas. Archivo adicional 1:La figura S1 corresponde a Rumex hymenosepalus espectro de absorción de solución acuosa. Se observa una banda característica centrada en 278 nm, asociada a las transiciones electrónicas de los anillos aromáticos conjugados con los grupos carbonilo de compuestos polifenólicos [82]. Esta banda de absorción en los espectros UV-Vis de las nanopartículas indica que los productos finales contienen moléculas de extracto que permanecen en ellos, Rivero-Cruz et. Alabama. han informado cuatro estilbenoides, dos flavan-3-oles y tres antraquinonas aisladas de R. himenosepalo [83] y Rodríguez-León et. Alabama. han realizado un estudio de resonancia magnética nuclear para determinar que Rumex hymenosepalus contienen moléculas importantes como el glucósido de estilbeno y el galato de epicatequina y el galato de epigalocatequina [61]. Estas moléculas participan como agentes reductores en la síntesis de nanopartículas. El proceso implica la desprotonación de algunos grupos -OH de anillos fenólicos para formar grupos =CO. Las moléculas polifenólicas se oxidan y los electrones liberados se transfieren a Ag ⁺ y Ag ³⁺ iones para formar Au ⁰ y Ag ⁰ .

La Figura 1b muestra los valores de los potenciales Zeta correspondientes a AuNP, AgNP y Au @ AgNP a concentraciones de 1, 10, 50 y 100 µg / mL. Las nanopartículas se dispersan en agua ultrapura y la medición se realizó por triplicado a 25 ° C. En general, en todo el rango de concentración, las nanopartículas exhiben valores de potencial zeta más negativos que -30 mV, alcanzando valores de -40 mV a una concentración de 100 µg / mL para AuNPs y Au @ AgNPs y -38 mV para AgNPs. Estos valores de potencial Zeta altamente negativos indican que las nanopartículas experimentan interacciones repulsivas entre ellas que evitan su agregación y permiten la estabilidad a largo plazo de los coloides metálicos [84,85,86]. Al correlacionar los resultados de la espectroscopía UV-Vis con los valores de potencial Zeta obtenidos, podemos establecer que los valores altamente negativos pueden deberse a la formación de complejos de moléculas polifenólicas del extracto en la superficie de la nanopartícula [87].

Para aplicaciones biológicas, es valioso obtener poblaciones de nanopartículas con tamaños monodispersos [88], por lo que la Fig. 1c yd muestra los valores DLS correspondientes al diámetro medio y al índice de polidispersidad (PDI) para AuNP, AgNP y Au @ AgNP a concentraciones de 1, 10, 50 y 100 µg / mL. El diámetro medio de Au @ AgNPs es de alrededor de 250 nm y se mantiene constante en función de la concentración, de manera similar para nanopartículas monometálicas con un diámetro medio alrededor de 122 y 135 nm para AuNPs y AgNPs, respectivamente. En el mismo caso observe que los valores PDI son alrededor de 0.3 para nanopartículas monometálicas y 0.2 para Au @ AgNPs. Estos resultados, donde los tamaños no varían con la concentración, indican que los diferentes sistemas de nanopartículas tienen buena estabilidad presentando una polidispersidad de tamaños moderada (0.3 ≥ PDI ≥ 0.2).

Se calculó la banda prohibida óptica del gráfico Tauc (archivo adicional 1:Figura S2). La banda de conducción de los materiales semiconductores tiene valores E _g <3 eV, como germanio de referencia tienen E _g <0,7 eV y el silicio es E _g =1,1 eV [89]. En nuestro caso, la banda prohibida para Au @ AgNP, AuNP y AgNP es 1,93 eV, 2,03 eV y 2,33 eV, respectivamente. Esto significa que estos materiales son considerados como semiconductores, y esta característica se obtuvo por los efectos de confinamiento cuántico que producen un aumento de la brecha energética, por lo que los nanomateriales pueden usarse como sensores, baterías y dispositivos optoelectrónicos [64].

Para establecer si los compuestos orgánicos están presentes en las nanopartículas, estos nanomateriales se caracterizaron por XPS y FTIR. La Figura 2a muestra los espectros de estudio del extracto vegetal y las nanopartículas. Como puede verse, todos los sistemas de nanopartículas presentan las señales características asociadas a los elementos carbono (C 1 s, 284,6 eV) y oxígeno (O 1 s, 532,2 eV) del Rumex hymenosepalus extraer. Este resultado confirma que las moléculas orgánicas del extracto están presentes en los nanomateriales obtenidos. Además, se obtuvieron espectros XPS de alta resolución para analizar los estados de oxidación de la plata y el oro en las nanopartículas (Archivo adicional 1:Figura S3). Para AgNPs (archivo adicional 1:Figura S3A), las señales en energías de enlace de 373,76 eV y 367,76 eV (∆BE =6,0 eV) correspondientes a la línea espectral 3d3 / 2 y 3d5 / 2 se pueden asociar con Ag ⁰ (plata metálica). Para AuNPs (archivo adicional 1:Figura S3B), el pico asociado con el acoplamiento espín-orbital 4f5 / 2 se encuentra en la energía de enlace 87,65 eV. Para 4f7 / 2, el pico de acoplamiento espín-orbital se encuentra en 83,98 eV. La relación de intensidades (I4f7 / 2> I4f5 / 2), ubicación y separación entre picos (ΔBE =3.67 eV) confirman que los iones de oro (Au ³⁺ ) se reducen completamente a oro metálico Au ⁰ [90]. Para Au @ AgNP, los espectros XPS de alta resolución correspondientes a la plata y el oro se muestran en el archivo adicional 1:Figura S3C y Figura S3-D, respectivamente. Estos espectros muestran el mismo comportamiento que sus homólogos monometálicos. Esto indica que tanto la plata como el oro son de valencia cero en la presentación core @ shell.

Espectros de levantamiento XPS ( a ) y FTIR ( b ) para Rh, Ag, Au y Au @ AgNPs

FTIR en la Fig. 2e muestra a 3296 cm ⁻¹ grupos hidroxilo, 2974 cm ⁻¹ (C – H) enlace del anillo aromático, 1689 cm ⁻¹ asociado con el estiramiento del grupo carbonilo (C =O) y 1689-1400 cm ⁻¹ debido al enlace carboxilato (C – O) y al estiramiento del enlace carbono-carbono y 1212 cm ⁻¹ asociado con el estiramiento de fenol C – O conjugado con AuNP, 1049 cm ⁻¹ Modo de estiramiento C – O de las moléculas del grupo éster de catequina [88,89,90] y 799 cm ⁻¹ la flexión fuera del plano en el fenol, la flexión C – H se informó en 964,4 y 829,39 cm ⁻¹ característica del resveratrol [91] desplazada en el extracto de Rh a 1031 y 867 cm ⁻¹ , respectivamente, y AuNP, AgNP y Au @ AgNP también se modifican, lo que es indicativo de la complejación de moléculas polifenólicas del extracto de Rh con nanopartículas.

La Figura 3a corresponde a una micrografía de STEM de campo claro representativa del sistema Au @ AgNPs a bajo aumento (barra de escala de 100 nm). A set of nanoparticles without agglomeration and with mostly quasi-spherical geometry can be seen. The same region is shown in dark field (HAADF) in Fig. 3b, and the core@shell structure can be observed, where can we distinguish Au-core looks more intense than Ag-shell, due to the difference in atomic number. Figure 3c and d corresponds to STEM higher magnification micrograph (scale bare 20 nm) of a nanoparticles group of system core@shell in a bright and dark field, respectively. Can be appreciated with clarity brilliant Au core and Ag shell lightly contrasted. These images show that the thickness of the Ag-shell varies between 3 and 5 nm. Additional file 1:Figure S4 corresponds to an individual images gallery where can be observed uniformity of Ag-shell.

Scanning Transmission Electron Microscopy at low magnification (scale bar 100 nm) of Au@AgNPs in Bright Field (a ) and HAADF (b ). A Small group of Au@AgNPs at higher magnification (scale bar 20 nm) in Bright Field (c ) and HAADF (d )

Figure 4a, c, and e corresponds to micrograph TEM of representative nanoparticles systems AuNPs, AgNPs, and Au@AgNPs, respectively. In all cases, nanoparticles have sphere-like morphology and are shown well separated from each other. This can be explained by the extract molecules onto nanoparticle surfaces, acting as spacers between them. Figure 4b, d, and f shows histograms corresponding to size distribution obtained by TEM and performed with 500 nanoparticles collected from 15 to 20 micrographs for each nanomaterial. The histogram presents Gaussian distribution with a mean size of 24 ± 4 nm (AuNPs), 13 ± 3 nm (AgNPs), and 36 ± 11 nm (Au@AgNPs). The discrepancy in values between DLS and TEM measurements with size distribution graph is due to conditions micro-environmental around the nanoparticles, while DLS shows a diameter for a system that includes metal, hydrated coating, and solvent by comparison in TEM measurements is a dry system where measurement is over metal only, in particular, in DLS the molecules used for complexation of nanoparticles (reducing agents and stabilizers) are dispersants that induce errors in sizing measurement and shifts it results to higher values [91].

TEM and size distribution of AuNPs in (a ) y ( b ), AgNPs in (c ) y ( d ), and Au@AgNPs in (e ) y ( f )

Figure 5 corresponds to the Au@AgNPs HAADF-STEM micrographics. A single nanoparticle is shown in Fig. 5a with a gold nucleus and silver cover perfectly delimited, the atomic number (Z) changes through the interface Au@Ag, intensity variations can be quantified by HAADF-STEM [67].

Au@AgNPs HRTEM (a ). Magnification from (a ) of interface core–shell (b ). FFT plot with Miller index (c ) and integrated image from FFT (Inverse) with interplanar distance (d )

The red square region is amplified to obtain an HRTEM micrography of the shell portion (Fig. 5b), and then to verify the crystalline shell structure, the nanoparticle periphery region was analyzed (discontinued square) with the Digital Micrograph 3.0 software (Gatan). Fast Fourier Transform (FFT) image of the selected area was obtained (Fig. 5c). Using the Inverse Fast Fourier Transform was possible to estimate interplanar distances of 2.3 Å, 2.0 Å, and 1.4 Å in Fig. 5d. These distances can be assigned, respectively, to the crystalline planes (111), (200), and (220) of face-centered cubic (fcc) silver according to Inorganic Crystal Structure Database (ICSD) at the FIZ Karlsruhe–Leibniz Institute for Information Infrastructure, Germany or the electron crystallography software Jems (V 4-5430, JEMS-SAAS, Switzerland) [92]. A similar analysis of crystal structure by HRTEM was carried out for monometallic nanoparticles as illustrated in Additional file 1:Figures S5 (for AuNPs) and S6 (AgNPs). In both cases, crystal structure corresponds to face-centered cubic (fcc).

EDS chemical analysis shows the presence of both metals for a group of bimetallic Au@AgNPs observed by TEM (Fig. 6a) in proportions of the atomic weight percent 77% of Ag (shell) and 23% of Au (cores) (Fig. 6b). In comparison, a single bimetallic (Fig. 6c) Au@AgNPs has proportions around 80% of Ag (shell) and 20% of Au (core) (Fig. 6d). To estimate the gold and silver atomic percentage on core@shell nanoparticles (Au@Ag), a quasi-spherical geometry approximation of nanoparticles morphology was considered. The AuNPs average diameter obtained from TEM size distribution (\(\overline{D}_{{{\text{Au}}}} =24\;{\text{nm}}\)) was used for core volume estimation (V _Au ), and Au@AgNPs average diameter \(\left( {\overline{D}_{{{\text{Au}}@{\text{Ag}}}} =36\,{\text{nm}}} \right)\) for core@shell volume estimation \((V_{{{\text{Au}}@{\text{Ag}}}} )\). So, shell volume is determined as \(V_{{{\text{Ag}}}} =V_{{{\text{Au}}@{\text{Ag}}}} - V_{{{\text{Au}}}}\). Total atomic content of Au and Ag was calculated considering an fcc crystalline structure (4 atoms per unit cell) for booth metals where the Au and Ag lattice parameters are 4.0783 Å and 4.0862 Å, respectively [93]. Atomic content estimation by this procedure is 70% for Ag (Shell) and 30% Au (Core), which differs by 10% concerning the measurement obtained by EDS. Figure 7 shows a theoretical estimation of silver and gold content and how varies as the thickness of the shell increases and the size of the core is kept constant (\(\overline{D}_{{{\text{Au}}}} =24\;{\text{nm}}\)). It is observed that for Au@AgNPs with a diameter greater than 30.2 nm, the atomic content of silver exceeds the content of gold. In Supplementary Material, a detailed description of calculation is carried out to obtain atomic contents percentage. Similar estimations of atomic percentages were carried out considering other Au-core diameters and varying Au@AgNPs diameter (Additional file 1:Figure S7A-B).

TEM and EDS of an Au@AgNPs group (a , b ) with Au 23 and Ag 77% at. and single Au@AgNPs (c , d ) with Au 20 and Ag 80% at

Variations of atomic content percentage versus increase thickness shell, where diameter AuNPs (24 nm) was kept constant

Figure 8 corresponds to XRD patterns for AuNPs and AgNPs as well as bimetallic Au@AgNPs. All the synthesized products have fcc crystalline structure as previously reported in the characterization by electron microscopy. Peaks for Au@AgNPs are located at 2θ diffraction angles of 38.25°, 44.4°, 64.9°, 77.85°, and 81.25°. As can be appreciated in the figure, the AgNPs and AuNPs diffraction peaks are found in the same positions mentioned with a difference of ± 0.5°. This is because Au and Ag have very similar lattice constants, so their diffraction patterns for fcc crystal structure are almost identical [94,95,96]. In this way, the diffraction peaks in Fig. 8 are assigned, respectively, to the crystalline planes (111), (200), (220), (311), and (222) of the gold and silver fcc structure by JCPDS:4-0783 and 4-0784 [97].

X-ray Diffraction AuNPs (pink), AgNPs (brown), and Au@AgNPs (red)

Antimicrobial Activity

Monometallic (AgNPs, AuNPs) and bimetallic (Au@AgNPs) materials were tested at four different concentrations:1, 10, 50, and 100 μg/mL. Selected microorganisms to evaluate antimicrobial activity were yeast Candida albicans , Gram-positive bacteria S.aureus , and Gram-negative bacteria E. coli . Growth kinetics curves in a time-lapse of 24 h are shown in Fig. 9.

The growth curves using AuNPs, AgNPs, and Au@AgNPs like inhibitory treatments on Candida albicans ( a - c ), Escherichia coli ( d - f ), and Staphylococcus aureus (g - yo )

Candida albicans

AuNPs show no effect on growth kinetics until 10 h (Fig. 9a), varying in a dose-dependent manner the absorbance reached at 24 h. Interestingly, with 50 µg/mL or more, the growth kinetic shows a steep negative slope from 10 h until reaching a 45% reduction at 24 h, which suggests an antifungal effect of these materials. This can be attributed to the ability of gold nanoparticles to interact with relevant proteins present in fungus such as H ⁺ -ATPase, affecting proton pump activity. This atrophying the ability of yeast to incorporate nutrients causing its death [61]. In Fig. 9b and c was observed that AgNPs and Au@AgNPs inhibit the growth of the yeast Candida albicans from 10 µg/mL. The determination of the MIC₅₀ concentration for both materials was estimated from the dose–response curve shown in Additional file 1:Figure S8. MIC₅₀ is defined as the concentration of nanoparticles that produces a 50% decrease in absorbance concerning the control (yeast without treatment). For AgNPs and Au@AgNPs, MIC₅₀ were 2.21 µg/mL and 2.37 µg/mL, respectively.

However, according to the EDS results (Fig. 9b), the silver content in Au@AgNPs is 64.85% mass. Thus, the concentration of silver in Au@AgNPs for MIC₅₀ is 1.53 µg/mL, 30% lower than in the case of AgNPs. Padmos et. Alabama. have demonstrated that silver nanoparticles possess important antibacterial features, but are cytotoxic by mammalian cells this effect has reduced using bimetallic nanoparticles especially using gold in the core of bimetallic nanoparticles [35].

Escherichia coli

In Fig. 9d, AuNPs do not show significant inhibition (< 15%) or affect the growth kinetics of E. coli . For AgNPs (Fig. 9e) at low concentrations, the Lag phase remains unchanged, but there is a marked decrease in growth ratio indicated by the slope decrement. At 50 µg/mL Lag phase lasts up to 16 h and viability reaches a maximum of 20% at 24 h. For 100 µg/mL, an apparent detachment of the growth phase of the microorganism is not observed. In Au@AgNPs (Fig. 9f), the first two concentrations do not show changes in their growth phase, but a phase delay of up to 2 h is observed compared to the control. It is interesting to note that the lag phase lasts up to 21 h for the 50 µg/mL concentration, finally there is no explicit growth behavior for the 100 µg/mL concentration.

Staphylococcus aureus

A comparative analysis of lag phase regrowth occurred after 12 h for Au (Fig. 9g), Ag (Fig. 9h), and Au@AgNPs (Fig. 9i) in the case of S. aureus at 50 µg/mL. For the highest concentration at 100 µg/mL, there is no growth of the bacteria. Additionally, we observe changes in the slope of respect control for Au, Ag, and Au@AgNPs at 1 and 10 µg/mL.

AuNPs interaction with these Gram-positive bacteria could be due to the charged surface that causes an electrostatic interaction, destabilizing membrane structure. Similar results, but with higher NPs concentrations, are reported for AuNPs synthesized using Ananas comosus fruit extract as reducing agent [98] and blue-green alga Spirulina platensis protein [99]. Yang y col. show MIC > 500 µg/mL for S. aureus (CMCC(B)26003), our AuNPs has shown inhibition with 10 times less concentration; in this case, a critical synergy exists with polyphenols molecules on coating and stabilizing the surface of nanoparticle [100]. ROS is generated of less to higher intensity [101] by AuNPs, polyphenols (plant extracts), and AgNPs, so AuNPs in synergy with resveratrol and epigallocatechin gallate (EGCG) promote antibacterial response over S. aureus [102] had the most feasible mechanism in this case. Penders et al. reported 250 and 500 µg/mL of AuNPs-like antibacterial agents over S. aureus increases in bacterial growth lag time and antibacterial effect [61, 98, 99].

We believe that inhibition is caused by AgNPs [103] accumulation and diffusion on bacteria related to NPs surface charges that promote electrostatic interactions [104] with the bacteria's membrane leading to higher penetration and damage. We think this is a similar mechanism described for interactions between E. coli biofilms and AgNps [105].

For Au@AgNPs, the obtained results are comparable to those reported by other workgroups [100, 101]; however, different authors suggest that the inhibition of the growth of the microorganisms is directly related to the thickness of the shell [100, 101]. Core–shell NPs showed low cytotoxicity when tested in NIH-3T3 fibroblasts cells (normal mammalian cells) [35]. A lower proportion of silver in the shell of the Au@AgNPs shows similar results to AgNPs [100], and Au core potentializes antibacterial effect, and minimizes the cytotoxicity.

Curves Growth Analyzed by the Modified Gompertz Model

To know how the growth ratios (µ ) and Lag phase (λ ) are quantitatively modified, the growth curves of microorganisms exposed to different concentrations of nanomaterials (Fig. 10) were adjusted by the Gompertz model (Eq. 2).

Kinetic parameters were obtained by the Gompertz model. Growth Rate µ and Time Lag Phase \(\lambda\) for S. aureus ( a , b ) and E. coli ( c , d )

In Fig. 10a, it can be seen that all nanomaterials produce a decrease in the replication rate of S. aureus populations when the concentration of nanoparticles increases. This effect results in slightly higher sensitivity for AuNPs. At a concentration of 50 µg/mL, the growth ratio is only 30% concerning control (Additional file 1:Tables S1-S24); at a concentration of 100 µg/mL, all materials inhibit the growth of the S. aureus población. The behavior of the adaptive phase for S. aureus with the different treatments is shown in Fig. 10b. It is observed that there are no significant differences in the material used, and at 50 µg/mL, the Lag phase has increased by almost 5 times compared to the adaptive phase of S. aureus (Additional file 1:Tables S1-S24). In general, we can establish that the different nanomaterials evaluated in S. aureus reduce the replication rate and postpone the adaptive phase in a dose-dependent manner until its inhibition at 100 µg/mL.

Figure 10c clearly shows that AuNPs do not affect the growth ratio µ of E. coli bacterias. Meanwhile, AgNPs produce a decrease over µ , reaching a minimum value corresponding to 19% to the control (µ for E.coli without treatment) for 50 µg/mL (see Additional file 1:Table S52). In contrast, Au@AgNPs completely inhibit the E. coli growth at 100 µg/mL. Analysis of the behavior of E. coli Lag phase exposed to different materials is shown in Fig. 10d. In this case, unlike Fig. 10b, each material has a characteristic response. Thus, AuNPs do not generate any modification in the adaptive phase of E. coli , while AgNPs and Au@AgNPs have a dose-dependent effect on the Lag phase, the latter material standing out. Thus, we can establish that AuNPs have no appreciable effect on E. coli bacteria, and Au@AgNPs can inhibit replication and, therefore, indefinitely postpone the Lag phase of E. coli . Interestingly, this effect is not achieved for AgNPs even though the net silver content is higher than in Au@AgNPs. This suggests that the core@shell presentation of both metals produces a synergy that favors antimicrobial activity. Feng et. Alabama. have reported an electron compensation phenomena from Au to Ag in core–shell and alloy structures, which derive in enhance the cytotoxicity of nanoparticles but kept it the antibacterial properties, that means, a synergy between Au and Ag are assumed, due to observed differences between the monometallic and bimetallic materials [106], but more research is necessary.

Figure 11 shows the results of the direct count study of colonies of microorganisms exposed to nanomaterials. In general, the behavior of the microorganism populations reproduces the results obtained from the growth kinetics study (Fig. 7a, e, i). For example, in Fig. 11a, the population of microorganisms (represented logarithmically) decreases significantly only at 19 h where AuNPs have killed 92% of C. albicans . Interestingly, for nanoparticles containing silver (Fig. 11b, c) a more pronounced population decline is observed. Au@AgNPs system at 50 µg/mL, almost entirely inhibits S. aureus at 7 h (99.5% of bacteria killed) although microorganism reactivates its growth for later times. MBC determination was not possible to obtain for tested concentrations (Additional file 1:Figure S9-11), according to experimental observation higher concentrations are required to show this effect. However, for C. albicans , AgNPs showed an MBC value of 50 µg/mL (Additional file 1:Table S55).

Effect of nanoparticles over microorganisms growth C. albicans exposed to AuNPs (a ), E. coli exposed to AgNPs (b ), and S. aureus exposed to Au@AgNPs (c ). All microorganisms were exposed at 50 µg/mL nanoparticles concentration