Controlled release of silver nanoparticles contained in photo

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Controlled release of silver nanoparticles contained in photo-responsive nanogels Camilo A.S. Ballesteros, Juliana Cancino Bernardi, Daniel S. Corrêa, and Valtencir Zucolotto ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00366 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Bio Materials

Title: Controlled release of silver nanoparticles contained in photo-responsive nanogels

Camilo A. S. Ballesteros1,2, Juliana Cancino Bernardi1*, Daniel S. Correa2,Valtencir Zucolotto1. (1) Nanomedicine and Nanotoxicology Group (GNano), IFSC, USP, P.O. Box 369, 13566-590 São Carlos, São Paulo, Brazil (2) Nanotechnology National Laboratory for Agriculture (LNNA), Embrapa Instrumentação, P.O. Box 741, 13560-970, São Carlos, São Paulo, Brazil. *Corresponding author: [email protected] (Juliana Cancino Bernardi)

Abstract Smart nanomaterials can selectively respond to a stimulus and consequently be activated in specific conditions, as a result of their interaction with electromagnetic radiation, biomolecules or pH change. These nanomaterials are produced through distinct routes and can be used in artificial skin, drug delivery and other biomedical applications. Here we report on the fabrication of an antibacterial nanogel formed by aniline and chitosancontaining silver nanoparticles, with an average size of 78 ± 19 nm. The AgNps nanogels release was triggered by light at 405 nm. Specifically, the electronic energy vibration resulting from the interaction of the irradiation with the AgNps surface plasmon breaks the hydrogen bonds of the nanogels and releases AgNps. To understand the perturbation of AgNps-nanogels against bacteria, membrane models studies were performed using the main components of cell membrane of Escherichia coli (E. coli),1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-racglycerol) (DPPG) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE). DPPG has more influence on the incorporation of the nanoparticles on the cell membrane due to the electrostatic interaction between the nanoparticles surface and lipid charged groups. The results indicate new possibilities for designing smart antibacterial photo-responsive nanogels with enhanced optical and antibacterial properties to increase E. coli death.

Keywords: Photoactivated Release; Silver Nanoparticles; Nanogels; Membrane Models.

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Introduction For many years antimicrobial drugs have been used to inhibit or kill microbes1–3. However, microbial resistance to these drugs has been reported recently, which reduces their effectiveness and causes severe public health problems4. One of the most promising strategies for fighting against bacterial resistance is the use of smart nanomaterials, once they can release their active molecules in a slow and/or in a controlled manner5– 8.These

nanomaterials can be activated in specific conditions upon interaction with electromagnetic radiation,

biomolecules, metallic and polymeric materials, as well as under pH change9–11. For example, nanogels can encapsulate several substances within their reservoirs and release it on demand at a later stage12–14. For that purpose, challenges need to be overcome to produce reliable and uniform nanogels whose properties can be altered by applying a remote stimulus, such as irradiation15,16. Nanogels loaded with metallic nanoparticles can be irradiated with specific wavelength, generating local electronic vibration, which leads to changes in the polymeric structure, releasing the nanoparticles to the surrounding environment17–19. Among nanoparticles with antibacterial properties, silver nanoparticles stand out, once silver ions released from the crystalline core can produce a chemical disequilibrium in the bacterial cell20–22. In addition, the nanogel degradation yields to non-toxic products for the environment, which is a fundamental prerequisite to biomedical applications23,24. Other applications show that multifunctional nanogels may be used as a general platform for therapeutic delivery; for example, in the study of stability, drug release behavior, and anticancer cytotoxicity25. For the application of antibacterial nanogels or therapeutic nanogels, it is crucial the design of smart nanomaterials with controllable properties to mediate specific biological response and consecutively understand the properties of biomaterials on biological functions at different level (e.g., cells, tissues, organs and the whole organism)26–28. To understand the mechanism of action of smart nanomaterials through cells, it is important to determine their behavior with the cell membrane. For such investigation, the Langmuir technique is a viable strategy, once it enables the control of phospholipids monolayers composition using the lipids presented in the cell membrane29. These mimetic membrane systems have been used to investigate the interaction between nanomaterials and membranes30. By using the Langmuir technique, it is possible to infer about the nanomaterials organization in the subphase, as well as the changes induced by them in the surface structure31. Consequently, this process allows inferring about the possible toxic effects arising from the interaction between the nanomaterials and the membrane32. Herein, the AgNps-nanogel was synthesized by the cross-linking between aniline and chitosan (CS), yielding hybrid polymeric nanogels, as illustrated in Fig. 1 (d). AgNps-nanogels exhibited optimal stability at pH from 3.0 to 7.4, with a surface plasmon band centered at 405 nm. The design of our AgNps-nanogel with 2 ACS Paragon Plus Environment

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materials such as chitosan and aniline gives more stability in the encapsulation of the AgNps by the crosslinking between them. The physical stimulus due to the irradiation at 405 nm excites the silver nanoparticles making possible the rupture of cross-linking and propitiating its own liberation along time. In this context, the kinetics of AgNps release under irradiation with a 405 nm diode LED was investigated, therefore, minimum inhibitory concentration (MIC) of the AgNps-nanogel dispersion was determined for gram-negative E. coli. The release profiles of the AgNps nanogels were studied using membrane models by the Langmuir technique, using phospholipids1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and 1,2-dimyristoyl-sn-glycero3-phosphoethanolamine (DMPE), which are the main components of inter plasmatic membrane of E. coli33–35.

Results and discussion Characterization of AgNps-nanogels UV-Vis absorbance spectrum of the AgNps-nanogels shows a surface plasmon resonance (SPR) of AgNps at 405 nm. A single band observed at 270 nm (Figure 1a) is due to the π-π* transition of benzene rings and excitation of the quinoid rings of aniline36. The characteristic surface plasmon absorption band of AgNpsnanogel reveals the presence of AgNps. Nanogel formation originated from the cross-linking between the -NH3+ protonated groups of aniline and OH- groups of CS37,38. The solubilization of CS in acetic acid solution results in an increase in the protonation level of CS39. The protonated amine group (-NH3+) in CS causes electrostatic repulsion and acts as a chelating agent that forces the silver ions to chelate and form AgNps inside of the nanogel. According to the inset of Figure 1b, the nanogels have a dimension of 78 ± 19 nm, value that is comparable to the size determined by dynamic light scattering (DLS) (79 nm and polydispersity index (PdI) of 0.28), indicating a narrow particle size distribution. Zeta potential is another important characteristic for nanoparticles, which value of + 39 mV was determined for our system, indicating a positive charge to the AgNps-nanogels originated from the amine groups from aniline and good suspension stability. The morphology of AgNps-nanogels is shown in the inset on Figure 1b (FESEM image), which evidences that AgNps are within the chitosan-aniline nanogel. This nanostructure can be classified as a nanogel structure, since the nanosystem formation is produced by the cross-linking between chitosan and aniline, while the AgNps is localized inside of the polymeric network37,38,40,41. The size of AgNps inside the nanogel (inset from Figure 1b) was estimated using ImageJ software as 18 ± 3 nm, which is in accordance with results from Figure S1 (support information SI) that shows the FESEM images of AgNps-CS nanoparticles, with average size of 17 ± 3 nm. The results allow estimating that there is a minimum of 5 AgNps to every single AgNps-nanogel system. 3 ACS Paragon Plus Environment


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Figure 1- a) UV-Vis absorption spectra of the AgNps-nanogel, displaying the surface plasmon resonance (SPR) at 405 nm, while the band at 270 nm originates from the π-π* transition of benzene rings. b) DLS of AgNps-nanogels in pH 7.2 with a size distribution of 79 nm and PdI 0.28 and FESEM images of AgNps-nanogels with average size of 78 ± 19 nm. The zeta potential was found at + 39 mV. The nanoparticle size inside of nanogel was determined by ImageJ software with an average size of 18 ± 3 nm. (c) FT-IR spectra showed the formation of AgNps after chelation inside polymeric network of CS in both cases AgNps-nanogels (i) and AgNps-CS (ii). (d) Illustration of AgNps-nanogels formation.

Nanogels without AgNps were produced (as synthesis control) using the same proportion of chitosan and aniline and the same concentration of reducing agent, yielding nanogels with larger size and wider size distribution (740 ± 260 nm) as displayed in FESEM images of Figure S2 (SI). Here, the protonated amine group (-NH3+) in CS molecules generates repulsion between them, increasing the diameter of the nanogel. The average size and stability of AgNps-nanogels in function of pH was investigated, as presented in Figure S3. The results showed that changes in pH modify the molecular conformation; for example, the nanogels are stable from pH 3 to pH 7.5, because the protonated amine groups in aniline attach to the hydroxyl groups of CS, leading the formation of nanogels. On the other hand, in alkaline pH, the interactions between functional groups 4 ACS Paragon Plus Environment

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of CS and aniline are affected by deprotonation of amine groups, which changes the morphological conformation of AgNps-nanogels, leading to aggregation, as observed by the increase of size values in the Figure S3a. The stability of the AgNps-nanogels was investigated during 10 days via DLS measurements at each 12 h, at pH 3 and 7. From Figure S3, one may observe a variation on size distribution and polydispersivity after 10 days of storage, indicating some instability for the system due to pH42,43 FT-IR spectra were collected to elucidate the interactions between the functional groups of the AgNps-nanogels components. The interaction between protonated amines and hydroxyl groups of the aniline-CS was verified in the nanogel formation. The development of AgNps inside the nanogels was observed in the protonated amine group of CS. In Figure 1c, the FTIR spectra of AgNps-nanogel and AgNps-CS are displayed. Changes in the band at 1392 cm-1 were observed, which corresponds to protonated amine –N-H in CS after chelation with silver ions to form AgNps inside of the nanogel13,14,44. Control FT-IR spectra were collected to elucidate the interactions between the functional groups of the AgNps-nanogel components, as observed in Figure S4. The interaction between protonated amines and hydroxyl groups of the aniline-CS was verified in the nanogel formation. The FT-IR spectra of AgNps-CS and CS are shown in Figure S4-A. The bands at 1381, 1334 and 654 cm-1 correspond to the stretching groups –C-Hand -N-H- in CS after silver ions chelation45–47. Figure S4-B demonstrates the cross-linking of aniline-chitosan in the formation of nanogel. Bands at 3349, 2810, 2599 and 2025 cm-1 are found in CS and aniline spectra14,48. This behavior could indicate that hydrogen bonds are formed between the protonated amine of CS-aniline and hydroxyl groups of CS. In the same spectra, the two bands at 1591 and 1497 cm-1 for AgNps-nanogel are assigned to the quinoid ring and benzene ring of aniline. Figure S4-C show the FTIR spectra of AgNps-nanogel and AgNps-CS. It is observed a deformation and stretching at 1392 cm-1 which suggests the protonation of amine in CS after those chelate with silver ions to form AgNps inside to the nanogel.

Minimum inhibitory concentration

To determine the minimum inhibitory concentration of AgNps-nanogels, the experiments with and without LED excitation over E. coli were carried out as described in experimental section. The bacterial concentration as a function of nanoparticles concentration is displayed in Figure 2. It is observed that irradiated samples present a lower concentration of bacteria compared to non-irradiated samples, which is due to release of AgNps under illumination, increasing the bacterial death. The minimum inhibition concentration (MIC) for the two experiments was determined as 0.9 µg/mL to 1-5×104 cfu/mL49. According with Wen-ru Li

a

concentration of 10 µg/mL completely inhibited the growth of 107 cfu/mL E. coli, since AgNps produce the 5 ACS Paragon Plus Environment


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leakage of sugars and proteins, inducing the respiratory chain dehydrogenases into an inactive state50,51.On the other hand, MIC values of 0.25 µg/mL and 6.25 µg/mL have been reported against E. coli after treated with AgNps52,53. This value is quite dependent of the particles coating and bacteria concentration.

Figure 2– Bacterial growth as a function of AgNPs-nanogels concentration for samples with and without exposition to LED excitation at 405 nm. MIC was determined as 0.9 µg/mL.

Some factors should be considered for this apparent lower toxicity, including coatings, increased dissolution of NP in contact with the bacteria surface, and underestimation of dissolved Ag+. Chitosan and aniline coatings influenced the oxidation pathways, and consequently the silver ion release. The control of the release pathway due to the surface coating justify why our system do not present a high effect to bacteria as expected by Ag0 → Ag+ in the evaluated experimental time, even with light exposition. Indeed, blue light is associated with AgNps-nanogel to release AgNps, and it can induce the formation of toxic reactive oxygen species that cause photochemical damage, making the system work in a bactericidal synergic effect at the molecular level. However, even 0.9 µg/mL does not diminish the bacteria growth results in a high extension at the time used in this experiment; it should be considered the molecular level effects that were highlighted by Langmuir monolayer results. Thus, 0.9 µg/mL was chosen for our investigation regarding membrane model experiments as a way to mimic the concentration that damages the membrane of E. coli.

Kinetic of release of AgNps

To investigate the interactions of the AgNps-nanogel with the membrane models, we first studied the release time of AgNps from the nanogel by applying LED light at 405 nm. The release of AgNps was 6 ACS Paragon Plus Environment

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determined by UV-Vis absorption spectroscopy, monitoring the absorption at 405 nm, which represents the SPR band of the AgNps. The LED light was applied at every 30 s during 600 s on a dispersion of AgNps-nanogel in a concentration of 0.9 µg/mL. After excitation with the LED, an increase of intensity in the plasmon band of the AgNps can be observed by UV-Vis absorption spectra, as illustrated in Figure 3a. The release time of Ag nanoparticles was determined at 240 s, which represent the totally detachment of the nanoparticles from the nanogel, Figure 3b. The small red-shift for the SPR band is a result of the structural change in the polymeric nanogel (changes in the dielectric properties of the medium) after irradiation, while the increase in the intensity of the SPR band indicates that there is a disaggregation of the nanogels and release of AgNps54,55, Figure 3a. Dynamic light scattering shows the size distribution of the Ag nanoparticles after irradiation, with an average size of 18 nm and PdI of 0.48, as illustrated in the inset of Figure 3b.

Figure 3 -

Photoactivation of AgNps-nanogels, (a) UV-Vis absorption spectra regarding the kinetics of release of AgNps. (b) the kinetic of release shows a stabilization at 240 s where most of the nanoparticles are released. The inset shows the DLS size distribution of AgNps of 18 nm and PdI of 0.48 after irradiation.

Figures 3 (a) and (b) show the increase in the absorbance at 405 nm upon LED irradiation. The initial absorbance (t = 0 s) is due to the silver nanoparticles exposed on the nanogel surface. After irradiation at 405 nm, the nanoparticles are released due to the excitation of the plasmon band, which breaks the cross-linking of the nanogels. Kenichi Niikura developed a polymeric nanogel composed of gold nanoparticles where the crosslinking was broken under laser irradiation at 532 nm, and consequently the anti-cancer drugs were released56. In our case, the breakdown of cross-linking is given by the silver nanoparticles, which are also the optically active substance. 7 ACS Paragon Plus Environment


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Membrane models Langmuir films may be advantageous over other membrane models, especially for investigating the molecular interactions occurring at the membrane surface in realtime57. There are experimental observations and theoretical studies of the similarity of monolayers with the arrangement of the membrane, whereby the Langmuir films occupied the same area with alignment and organization that biomolecules exhibit in the membrane cell57,58. However, Langmuir monolayers have some disadvantages because this model only permits the simulation of half of the cell membrane, which is deficient for investigations regarding molecular transport across the membrane31,59. The interaction of AgNps-nanogels with membrane models was investigated using DMPE and DPPG lipids as the components of the monolayers. This nanosystem has a particular behavior when it is irradiated by diode LED at 405 nm. In this case, the surface plasmon band of AgNps is excited by the irradiation, detaching this nanomaterial of the polymeric matrix or nanogels. By knowing this behavior, the interaction at the molecular level mimicking a membrane of E. coli bacteria is possible, once phospholipids are present in both the inner and the outer membranes. E. coli membranes are composed basically of zwitterionic lipids 75% PE, and a small portion of anionic lipids composed by 20% PG, and 5% CL, composition that is relatively constant under a broad spectrum of growth conditions61,62. Initially, we evaluated the interaction of AgNps-nanogels with DMPE and DPPG lipids individually, following the evaluation of the binary mixture using the same proportion found in bacteria63. This lipidic composition was chosen in terms of charge; specifically, we intend to explore the interaction of the polar headgroup of the lipids molecules and the nanogels, before and after the light exposure. The concentration of AgNps-nanogels used in this study was 0.9 µg/mL, as determined by MIC for E. coli. The isotherms were obtained for a DPPG monolayer on aqueous subphase in the absence and presence of the nanomaterials (Figure 4). The barrier velocity was maintained at 10 mm/min and the monolayer was subjected to 3 successive cycles of compression and decompression.


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Figure 4 - Evolution of the DPPG monolayer upon interaction with the nanosystem, applying or not LED light. (a) the kinetics show the variation in the surface pressure vs time related to the interaction between the components of the system, (b) surface pressure vs molecular area for DPPG monolayers in the presence of AgNps-nanogels, with and without illumination. (c) schematic diagram of the interaction between AgNps and DPPG before and after irradiation.

Figure 4a shows the surface pressure versus time of the DPPG monolayer containing AgNps-nanogels, with or without LED illumination for 240 s. It is possible to verify a significant variation in the surface pressure as a function of time when the LED excitation is applied, with the surface pressure increasing from 8 to 11 mN/m. The latter may be related to the detachment of the AgNps from the polymeric network, increasing the surface pressure. The increase of the surface pressure is exclusively due to the interaction between nanogels and DPPG lipid monolayer, once control experiments from the AgNps release did not affect the surface activity of the system. These results were supported by the isotherms from AgNps-nanogels and DPPG system itself that did not present any surface activity. Large variation in the molecular area is observed in the isotherms from Figure 4b. When the DPPG monolayer is compressed at a surface pressure of Π = 30 mN/m, a molecular area of 65 Å2/molecule is observed. In contrast, an increase in the molecular area of 110 Å2/molecule is observed in the presence of AgNps-nanogel, and this value increased even more when the LED light is applied (130 Å2/molecule) for a same surface pressure. This behavior suggests that the DPPG monolayer is affected by the AgNps release in the subphase, with a possible incorporation of the AgNps into the lipidic monolayer (Figure 4b), causing perturbation in the monolayer, compared to the packed monolayer without the AgNps-nanogels 9 ACS Paragon Plus Environment


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(Figure 4c). However, the movement of the barriers did not impair the interaction during compression and decompression of the monolayer, as observed in Figure S5a. The monolayer constituted by DMPE did not present significant differences in the surface pressure variation or in the molecular area of the isotherms upon addition of the AgNps-nanogels. Figure 5 shows the kinetics and the isotherms of the interaction between the DMPE lipids with the AgNps-nanogels.

Figure 5-

Evolution of the DMPE monolayer upon interaction with the nanosystem, applying or not LED light. (a) variation in the surface pressure vs time related to the interaction between the DMPE monolayer and nanosystem, (b) Surface pressure vs molecular area for DMPE monolayer in the presence of AgNps-Nanogels with and without LED illumination (c) schematic diagram of the interaction between AgNps and DMPE before and after irradiation.

Figure 5a shown an increase in the surface pressure values for DMPE monolayers with time, in the presence of AgNps-nanogels. However, this variation was smaller than that observed for the DPPG system. The isotherms from Figure 5b show that the DMPE monolayer lipid packaging was not changed, even after the application of the LED light. In this case, a small variation in the molecular area of nearly 8 Å2/molecule was observed at a surface pressure of 30 mN/m. The latter may be associated with the repulsive electrostatic effects between AgNps-nanogels (amine groups) and lipid molecules that are more positive at the worked pH due to the protonated amine group (Figure 5c). A decrease in the molecular area values in the decompression of the monolayer occurred as a function of the surface pressure, as shown in Figure S5b.


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After analyzing the interaction between AgNps-nanogels and the DPPG and DMPE lipids individually, we investigated the effects of the nanomaterials in a mimicked monolayer composed by a binary mixture of DPPG and DMPE. DMPE was solubilized in chloroform and DPPG in methanol:chloroform at a concentration of 1 µmol/L. The binary mixture was prepared at a proportion of 1:4 (v/v). The results are shown in Figure 6.

Figure 6 - Evolution of the binary DPPG:DMPE (1:4 (v/v)) monolayer upon interaction with the nanosystem, applying or not LED light. (a) Variation in the surface pressure vs time related to the interaction between the binary DPPG:DMPE monolayer and the nanosystem. (b) Surface pressure vs molecular area for binary DPPG:DMPE monolayer in the presence of AgNpsnanogels with and without LED illumination (c) schematic diagram of the interaction between AgNps and the DPPG:DMPE before and after irradiation.

Figure 6a shows the surface pressure vs time of the binary monolayer containing DPPG:DMPE (1:4 (v/v)) and AgNps-nanogels, with and without exposition to LED light. It is possible to verify a variation of at least 2 mN/m in the surface pressure when the LED excitation is applied, which is similar to that observed for the DPPG system (Figure 4). In the isotherm, it is observed a variation in the area of at least 0.001m2 for a surface pressure of 30 mN/m. In contrast, when LED excitation is applied, for the same surface pressure, a small variation in the area of 5 cm2 is observed, Figure 6b. This behavior was expected, because of the contribution from the two lipids at the interface. The surface pressure values are highly influenced by the physico-chemical characteristics of the lipid molecules. The contribution of the DPPG molecules in the interaction with the nanogel is very strong, mainly due to the existence of electrostatic interaction between the charges of the lipids 11 ACS Paragon Plus Environment


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and the AgNps. The nanomaterials are located between the phospholipids (DPPG), breaking the structure of the membrane, Figure 6c.

Conclusions The synthesis, characterization and release profile of AgNps photoresponsive nanogels were investigated. The AgNps were chelated in the aniline-chitosan nanogels configuration, which cross-linking occurred between protonated amine groups of aniline and chitosan and OH- from chitosan. The presence of AgNps was confirmed by UV-Vis absorption spectrum, with a surface plasmon band at 405 nm as well by scanning electron microscopy. The nanomaterial exhibited a SPR band that can be stimulated by light at 405 nm. Upon irradiation, an increase in the kinetic energy of the system occurs, detaching the AgNps from the polymeric network. The irradiation to the AgNps-nanogels in the presence of E. coli indicated the bacterial death. The membrane models showed different behaviors for each type of lipid (DPPG, DMPE and DPPG:DMPE) when interacting with the AgNps-nanogel exposed or not to LED excitation. The monolayer composed by an anionic DPPG lipid was affected by the presence of the AgNps-nanogel in the subphase, indicating the incorporation of the Ag nanoparticles between the lipids. In addition, a less packed configuration was found under irradiation, compared to the monolayer without the nanogels. The monolayer constituted by DMPE (a zwitterionic lipid) did not present significant differences in the surface pressure variation. This effect can be associated with the repulsive electrostatic effect between the nanogels and the lipid molecules that are more positive at the worked pH. The binary monolayer composed of DPPG:DMPE showed similar behavior with the DPPG monolayer, which allows an electrostatic interaction between these molecules and the AgNps. The mechanism of action can be associated to the incorporation of the nanoparticles between DPPG molecules in the cell membrane. The results presented open new possibilities for designing smart antibacterial photo-responsive nanogels with superior properties.

Supporting Information Available: Additional information on materials characterization and results. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments


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The authors are grateful to FAPESP (grant numbers: 2012/03570-0, 2017/12174-4), CAPES, CNPq, MCTISisNano and Rede Agronano (EMBRAPA) from Brazil for financial support.

Experimental section

Materials

Chitosan (CS, medium molecular weight), Aniline Hydrochloride ≥ 99.0% (mw = 129.59 g/mol), Silver nitrate (mw = 169.87 g/mol) and Sodium Borohydrade granular, 99.99% (mw = 33.83 g/mol) were purchased from Sigma-Aldrich. Glacial Acetic Acid (CH3COOH) was purchased from Synth (Brazilian Industry, São Paulo, Brazil). All aqueous solutions were prepared with double-distilled water and the chemicals were used without further purification.

Synthesis of AgNps-nanogels using a complexation-reduction method The synthesis of the AgNps-nanogels was carried out through a complexation-reduction method adapted from the work reported by Fwu-Long Mi

64.

The main difference of our system compared to the Fwu-Long

studies is the use to external stimulation to control Ag+ release. The design of our AgNp-nanogel with materials such as chitosan and aniline provides more stability for the encapsulation of the AgNps by the cross-linking between them. The irradiation at 405 nm excites the silver nanoparticles, making possible the rupture of crosslinkings, allowing its own liberation along time. For the synthesis, chitosan (CS) (16.5 % w/v, 50 mL) was dissolved in acetic acid (1 % v/v, 50 mL). Aniline (A) hydrochloride (10 mM) were mixed with the CS solution and left under magnetic stirring for 1 h. The reaction produces cross-linking between aniline and chitosan due to the covalent character between the two molecules37,38,40,65. Following, AgNO3 (0.5 mM) was added to the solution (chitosan-aniline) and mixed during 1 h under magnetic stirring, until the resulting mixture appeared milky with greenish opalescence. Silver ions in the A-CS complex were reduced using NaBH4 (4.5 mM) at room temperature. Specifically, NaBH4 (100 mL) was added to the solution in intervals of 30 min (three times) to protonate the NH2 group in aniline to NH3+ and to enhance the hydrogen bonds to -OH- groups of chitosan. Silver ions in the A-CS complex nanogel chelate in AgNps, because the protonated amine (-NH3+) of CS inside the nanogel attracts the ions and permits the formation of AgNps. In the case of nanogels without AgNps, we assumed that protonated amine group (-NH3+) in CS molecules does not have the function of a chelating agent. 13 ACS Paragon Plus Environment


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This charge generates electrostatic repulsion, increasing the diameter of the nanogel64. The synthesized material was washed by centrifugation, first for 2 min to 10000 g to eliminate residues of the synthesis and a second time by 1 min, 10000 g.

Determination of AgNps-nanogels concentration To determine the concentration of AgNps-nanogels 1 mL of dispersion was poured on glass sheets (triplicate). The dispersion of AgNps-nanogels was then dried using a vacuum pump (TBV5-Tander). At the end of drying process, the concentration of AgNPs-nanogel was determined as 57.6 µg/mL.

Synthesis of Ag-Chitosan Nanoparticles (AgNps-CS)

AgNps-CS nanoparticles were prepared to understand how silver nanoparticles are release from AgNpsnanogels. The visualization of this effect can be assessed by observing the difference between silver nanoparticle loaded by chitosan (AgNps-CS) and silver nanoparticles nanogels (AgNps-nanogels) containing silver, chitosan and aniline. For preparing the AgNps-CS nanoparticles, CS (16.5 % w/v) was dissolved in acetic acid (1 % v/v, 25 mL). Then, AgNO3 (0.5 mM) was added to the solution, which was kept at room temperature for 10 min66. Subsequently, the reducing agent NaBH4 (4.5 mM, 100 mL) was added and the solution was kept under magnetic stirring during 1 h66. One drop of the dispersion of AgNps-CS was poured on silicon sheet and dried using vacuum pump without any type of coating for the field-emission scanning electron microscopy (FESEM) micrographs.

Kinetics of AgNPs release using a LED source at 405 nm The release kinetics of AgNps was studied using a diode LED (LHUV-0405) at 405 nm with an intensity of 32 mW/cm2. The diode LED is set 5 cm distant from the nanogels dispersion (0.9 µg/mL). For the measurements, 500 µL of AgNp-nanogels dispersion (0.9 µg/mL) was introduced in a glass cuvette and irradiated in intervals of 30 s, 60 s, 90 s, 120 s, up to a total of 600 s. In each interval a new sample was used; for example, 30 s a sample, then 60 s another sample and so on consecutively up to 600 s until a kinetic curve for the release of silver nanoparticles from the nanogel was obtained. The samples were monitored by UV-Vis absorption spectroscopy (405 nm) at room temperature, scheme S6. 14 ACS Paragon Plus Environment

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Kinetics of AgNPs release using excitation at 405 nm on the Langmuir trough

For the evaluation of the AgNps release from AgNps-nanogel in the Langmuir trough, a tool of aluminum box with diode LED was set over the Langmuir trough for irradiation of the subphase, as displayed in Figure S7. The diode led was maintained at a distance of 5 cm and applied light during 240 seconds.

Minimum inhibitory concentration (MIC) for Escherichia coli

For minimum inhibitory concentration (MIC) experiments, the AgNps-nanogels were dissolved in 100 µL of Mueller Hinton Broth to obtain a concentration of 57.6 µg/mL in the first well. Next, a two-fold dilutions series were carried out using a microliter plate until the 8th well reached a concentration of 0.45 µg/mL. 10 µL of the microorganism was added to a final concentration of 1-5×104 cfu/mL on each well. In our experiments, the effect of light irradiation on the 96-well plate was investigated as observed on Figure S8. Therefore, tests were carried out using a 96-well plate with and without irradiation. The growth of bacteria was hourly monitored by UV-Vis absorption spectroscopy (at 625 nm) during 12 h. This experiment was compared with the membrane models experiment to determine the effect of the AgNps-nanogels in the antibacterial mechanism (with and without light irradiation).

Membrane models

To develop the experiments, two phospholipids were chosen as the main components of the plasmatic membrane of E. coli: 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) a zwitterionic phospholipid, and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) an anionic phospholipid at pH 5.567,68. The chemical structures of both lipids are displayed in Figure S9. Both lipids were used in the concentration of 1 µmol/L, DMPE was solubilized in chloroform and DPPG in methanol:chloroform solution in the proportion of 1:4 (v/v). The variation of surface pressure was recorded as a function of area per molecule. The experiments were performed at 21 ± 1°C. The isotherm of area-surface pressure (Π-A) was obtained by dispersing 6 µL of the phospholipids in the water surface using a Hamilton microsyringe. These monolayers were left to equilibrate for 10 min before compression in the subphase without nanoparticles and for 40 min in the subphase containing 15 ACS Paragon Plus Environment


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AgNps-nanogels. In the compressed-decompressed experiments, the barriers were moved at a speed rate of 10 mm/min until the surface pressure reached 50 mN m−1 for three successive cycles. For the analysis of the interaction of nanomaterials, the unique change was the addition of the nanomaterials on the subphase and application of LED at 405 nm when necessary.

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Graphical Abstract


Controlled release of silver nanoparticles contained in photo

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