Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract.

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COLSUB-6582; No. of Pages 11

Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx

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Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract Luminita David a , Bianca Moldovan a , Adriana Vulcu b , Liliana Olenic b , Maria Perde-Schrepler c,∗∗ , Eva Fischer-Fodor c , Adrian Florea d , Maria Crisan e , Ioana Chiorean f , Simona Clichici g , Gabriela Adriana Filip g,∗ a

Faculty of Chemistry and Chemical Engineering, Babes-Bolyai University, 11 Arany Janos Street, RO 400028, Cluj-Napoca, Romania National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath Street, RO 400293, Cluj-Napoca, Romania c “Ion Chiricuta” Oncology Institute, 34-36 Republicii Street, 400015, Cluj-Napoca, Romania d Department of Cell and Molecular Biology, “Iuliu Hatieganu” University of Medicine and Pharmacy, 6 Louis Pasteur Street, 400349, Cluj Napoca, Romania e Histology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 13 Emil Isaac Street, 400023, Cluj-Napoca, Romania f Faculty of Mathematics and Computer Science, Babes-Bolyai University, 1 Kog˘alniceanu Street, 400084, Cluj-Napoca, Romania g Physiology Department, “Iuliu Hatieganu” University of Medicine and Pharmacy, 13 Emil Isaac Street, 400023, Cluj Napoca, Romania b

a r t i c l e

i n f o

Article history: Received 21 December 2013 Received in revised form 10 August 2014 Accepted 13 August 2014 Available online xxx Keywords: European black elderberry (Sambucus nigra) Adoxaceae family Silver nanoparticles Anti-inflammatory effect Psoriasis

a b s t r a c t This research aimed at reporting the synthesis, characterization and evaluation of the anti-inflammatory effects of some new biomaterials based on silver nanoparticles and polyphenols rich natural extracts. A fast and eco-friendly extracellular biosynthesis of silver nanoparticles (AgNPs), using European black elderberry (Sambucus nigra – SN, Adoxaceae family) fruit extracts was developed. The phytosynthesized nanoparticles exhibited an absorbance peak at 426 nm, characteristic for AgNPs and their sizes were ranged from 20 to 80 nm. The anti-inflammatory properties of AgNPs were assessed in vitro on HaCaT cells exposed to UVB radiation, in vivo on acute inflammation model and in humans on psoriasis lesions. In vitro, our results demonstrated the anti-inflammatory effects of functionalized AgNPs by the decrease of cytokines production induced by UVB irradiation. In vivo, the pre-administration of AgNPs reduced the edema and cytokines levels in the paw tissues, early after the induction of inflammation. The present study also demonstrated the possible use of synthesized AgNPs for the treatment of psoriasis lesions. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Nanostructured noble metals have gained much popularity lately, being used in several technological and medical applications such as molecular imaging [1] drug delivery [2], development of materials and medical devices for diagnosis and treatment [3,4]. Various methods have been employed to prepare metal nanoparticles, the most important being the chemical reduction, ultraviolet and microwave radiation and also photochemical and sonoelectrochemical methods [5]. Due to the extreme toxicity of chemicals, the development of an alternative eco-friendly, simple and reliable synthetic method based on the reducing capacity of some compounds from natural organisms became mandatory.

∗ Corresponding author. Tel.: +40 745268704. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Perde-Schrepler), gabriela.fi[email protected], adrianafi[email protected] (G.A. Filip).

The phytochemical synthesis of silver nanoparticles (AgNPs) using plants or fruit extracts plays an important role in the field of nanotechnology and nanomedicine as it offers alternative therapeutic options which are safe, free of side effects and effective for a wide variety of diseases. Several studies demonstrated that AgNPs possess remarkable inhibitory effect against microorganisms [6,7] as well as free radical scavenging and antiinflammatory properties [8,9]. Experimental data assigned AgNPs wound healing properties, antitumor [10,11], antiviral, antibacterial [12] and anti-angiogenetic effects [13]. It has been shown that AgNPs induce apoptosis and reduce the level of matrix metalloproteinase in wounds [14], inhibit angiogenesis [15] and vascular permeability through vascular endothelial growth factor (VEGF), interleukin (IL)-1␤ and advanced glycation end products in bovine retinal endothelial cells [10]. However, the effects of AgNPs on the inflammatory response have not been extensively investigated yet. Furthermore, some very recent studies have shown that silver nanoparticles exerts cytotoxic, pro-inflammatory and proapoptotic effects mediated via reactive oxygen species (ROS) [16] generated in normal [17] and tumor cell lines [18].

http://dx.doi.org/10.1016/j.colsurfb.2014.08.018 0927-7765/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: L. David, et al., Green synthesis, characterization and anti-inflammatory activity of silver nanoparticles using European black elderberry fruits extract, Colloids Surf. B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.08.018

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ARTICLE IN PRESS L. David et al. / Colloids and Surfaces B: Biointerfaces xxx (2014) xxx–xxx

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This paper focuses on exploiting European black elderberry recognized in Europe for its health-promoting properties for many generations. Its fruits have higher antioxidant capacity than vitamin C or E, are capable of enhancing immune system response through elevated production of cytokines, and have been used in European folk medicine to circumvent the ravages of colds, asthma, arthritis, and even constipation for thousands of years [19–21]. The natural compounds extracted from these polyphenols rich fruits were chosen to mediate the synthesis of AgNPs due to their well-known biological properties, such as antioxidant [22,23], antiinflammatory [24] and anti-cancer activity [25]. Although steroids and non-steroidal anti-inflammatory drugs (NSAIDs) are the main therapeutic agents in inflammation, they can cause serious side effects. Therefore, the development of new materials with comparable results and no side effects is needed. The purpose of our study was to synthesize silver nanoparticles using European black elderberry fruits extract, to characterize them and to evaluate their biological activity in several systems: HaCaT cells exposed to UVB radiation, carrageenan-induced paw edema in rats and psoriasis lesions in humans.

2.4. Characterization of the AgNPs UV–vis spectrum was recorded with a Shimadzu UV–Vis spectrophotometer ( = 300–850 nm). The morphology and size distribution of nanoparticles were examined by a TEM JEOL-JEM 1010 instrument (JEOL Inc). FTIR spectroscopy of green synthesized AgNPs was performed on a JASCO 6100 spectrophotometer (spectral domain 5000–500 cm−1 , the resolution was 4 cm−1 , with the sample as KBr pellets). The X-ray diffraction (XRD) analysis was assessed with D8 Advance diffractometer equipped with a germanium monochro˚ mator with CuK␣1 radiation ( = 15.4056 A). The nanoparticles have been analyzed by means of inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin Elmer ELAN DRC spectrometer. The Zeta potential of the biosynthesized AgNPs was performed on Malvern Zetasizer Nanoseries compact scattering spectrometer (Malvern Instruments Ltd.; Malvern, UK). 2.5. In vitro studies

2. Materials and methods 2.1. Vegetal material and preparation of the extracts Samples of European black elderberry fruits were harvested in September 2012 from Cluj-Napoca, Romania (Lat. 46◦ 45 N, Long. 23◦ 36 E). Plant with complete herbarium was identified and authenticated by Prof. Emeritus M. Tamas, Department of Botany, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy Cluj Napoca, Romania. Fruits were packed in polyethylene bags and kept frozen at −18 ◦ C before being subjected to polyphenols extraction. The frozen fruits were crushed in a mortar. Forty grams of fruits were transferred to an Erlenmeyer flask and 200 ml of food grade acetone and 50 ml double distilled water were added. The mixture was stirred for 1 h at room temperature and then filtered through Whatman no. 1 paper under vacuum. The filtrate was concentrated under vacuum until total removal of acetone. The total anthocyanins content was determined using the pH differential method [26] and the total phenolic content was assessed by the Folin–Ciocalteu method [27]. 2.2. Reagents Silver nitrate, NaOH and EDTA-Na2 were obtained from Merck (Germany) and absolute ethanol and n-butanol from Chimopar (Bucharest, Romania). ELISA tests and a multiplex cytokine kit were obtained from R&D Systems (Minneapolis, MN, USA). Glutaraldehyde for TEM was from EMS, (Hatfield, USA), osmium tetroxide from Sigma–Aldrich (St. Louis, USA), Epon 812 resin (Fluka GmbH, Buchs Switzerland). All other chemicals and reagents were purchased from Sigma–Aldrich (Germany) and were of high grade purity. 2.3. Synthesis of silver nanomaterials To 6.6 ml 1% AgNO3 solution 200 ml double distilled water were added. The boiling silver salt solution was mixed with 16.6 ml fruit extract (total anthocyanin content 24 × 10−3 mM) while vigorously stirring for 10 min, until a brown solution was obtained. The suspension has been preserved at 4 ◦ C. The colloids were stable for 1 month. Before usage, the silver nanoparticles suspension was centrifuged at 15,000 × g for 10 min. The supernatant was removed and double distilled water was added for further cleansing. The mixture was centrifuged at 15,000 × g for another 10 min to remove supernatant. The procedure was twice repeated.

2.5.1. Cell culture HaCaT, a normal, spontaneously immortalized keratinocytes cell line, was maintained in high glucose (4.5 g/L) DMEM, supplemented with 10% fetal calf serum, 1% l-glutamine and 1% Penicillin–Streptomycin. The cells were kept at 37 ◦ C in a humid incubator with 5% CO2 and observed daily at an inverted phase Zeiss AxioObserver D1 microscope. 2.5.2. Transmission electron microscopy HaCaT cells, both the control group and cells exposed to AgNPs, were processed for transmission electron microscopy (TEM). They were fixed for 1.5 h with 2.7% glutaraldehyde, washed four times with 0.1 M phosphate buffer (pH 7.4), postfixed for 1.5 h with 1.5% osmium tetroxide, dehydrated in an acetone series, and embedded in Epon 812 resin. The ultrathin sections obtained with glass knives using a Bromma 8800 ULTRATOME III (LKB, Stockholm, Sweden) were collected on 300 mesh copper grids (Agar Scientific Ltd., Stansted, UK). In order to visualize better the localization of AgNPs within the cells, the sections were not contrasted. The samples were examined on a JEOL JEM 1010 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV acceleration voltage, and photographed with a Mega VIEW III camera (Olympus, Soft Imaging System, Münster, Germany). 2.5.3. Cytotoxicity assays Cells were plated in 96-well plates, 20,000 cells/well. Treatments were done after 24 h with solutions containing various concentrations of nanoparticles (AgNPs) (950, 475, 190, 95, 47.5, 19 and 2.37 ␮g/ml) and European black elderberry fruits extract of various anthocyanin contents (554.3, 277.15, 110.86, 55.43, 27.7, 11.08, 5.54 mg/L), three wells for each concentration. For each experiment, at least three wells were left untreated (control). After 24 h, the cellular viability was assessed by MTT, a colorimetric cell viability assay developed by Mossmann, with modifications [28]. Absorbance was read on a Biotek Synergy 2 microplate reader, at 492 nm. Each individual experiment was repeated at least 3 times. 2.5.4. Inflammation assay HaCaT cells were seeded on 24 well plates. Two plates were used: one was kept in the dark, while the other one was exposed to UVB. Both plates were treated with 150 ␮l/well AgNPs (19 ␮g/ml) and European black elderberry fruits extract (110.86 ␮g/ml), for 30 min before their exposure to UVB. Irradiation was performed




in the culture plates (without lids) using a broadband UVB source Waldmann UV 181. The emitted dose was measured with a Variocontrol radiometer (Waldmann GmbH, Germany). The exposure dose was calculated using the formula: Dose (mJ/cm2 ) = Exposure time (76 s) × Intensity (1.31 mW/cm2 ) and was 100 mJ/cm2 . At 24 and 48 h after irradiation supernatants were collected and the inflammatory cytokines IL-1␣ and IL-6 were assessed by ELISA, using Quantikine kits from R&D, following the manufacturers’ protocol. Absorbance was read at 450 nm on a Tecan Sunrise reader. The corresponding concentrations were obtained on the basis of the standard absorption curve using the Magellan 3 software. 2.5.5. Model of carrageenan-induced inflammation in Wistar rats All procedures for in vivo experiments were approved by the Ethics Committee on Animal Welfare of the “Iuliu Hatieganu” University, in accordance with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes, Council of Europe No. 123, Strasbourg 1985. The animals were obtained from the Animal Facility of “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania and were acclimatized for a week under the following conditions: 12-h light/12-h dark cycle, 35% humidity, free access to water and fed by a normocaloric standard diet (VRF1). The rats were deprived of water only during the experiment to minimize variability in edematous response. Paw edema was induced in male Wistar rats (110–130 g, 3 months old) by the injection of 100 ␮l carrageenan 1% (␭-carrageenan, type IV) in the right hind foot pad [29]. The left hind foot pad was injected with the same volume of saline solution. The extract and colloidal suspension were orally administered daily, 4 days prior to injection of carrageenan, in a volume not exceeding 0.5 ml. Animals were randomly divided into 4 groups of 8 animals each: group 1, positive control group, was treated with Indomethacin (5 mg/b.w. in 0.5 ml carboxymethylcellulose); group 2-pre-treated with 0.9% NaCl solution as a negative control; group 3-pre-treated with 15 mg/b.w./day European black elderberry fruits extract; group 4-pre-treated with AgNPs 0.3 mg/b.w (0.19 mg/ml). Rat paw edema was assessed by the volume displacement method (plethysmometer Ugo Basile 37140, Italy) before the administration of the substances and at 2 h, 24 h and 48 h after carrageenan injection. Anti-inflammatory activity was evaluated for each animal in comparison with control and calculated using the formula: I % = [(1 − (dt/dc)] × 100 where dt is the difference in paw volume in the drug treated group and dc is the difference in paw volume in the control group [30]. Thereafter, animals were sacrificed and soft paw tissues were harvested and used for the inflammation quantification. 2.5.6. Preparation of tissue extracts The soft tissues were prepared in 50 mM TRIS–10 mM EDTA buffer (pH 7.4) and homogenized with a Polytron homogenizer (Brinkman Kinematica, Switzerland). The suspension was centrifuged for 5 min at 3000 × g and 4 ◦ C to prepare the cytosol fraction. The protein content in homogenates was measured with Bradford method [31]. Soft tissue homogenate from each group of animals was stored in aliquots at –80 ◦ C until assayed. 2.5.7. Evaluation of inflammation Three different cytokines (IL-1␣, IL-␤ and IL-6) were measured in rat paw homogenate with Luminex 200 (Luminex Corporation, Austin, TX, USA) using a multiplex cytokine kit, the assay being performed in accordance with the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). 2.5.8. Effect of AgNPs on psoriasis 2.5.8.1. Cream preparation. An oil-in-water cream containing AgNPs was prepared. A hot lipophilic phase obtained by combining

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petrolatum, vaseline oil and cetyl alcohol was emulsified in the aqueous phase, containing the AgNPs and mixed with a hydrophilic surfactant (polysorbate 60). The emulsion was homogenized at high speed (400 rpm) for at least 5 min, then homogenization was continued in an ice/water bath until the temperature of the cream was 25 ◦ C and a semi-solid preparate was obtained. The creams were used for the assessments of the AgNPs effects on the psoriatic lesions. The assessment of the skin lesions was performed using the noninvasive, high frequency ultrasound device Dermascan C 20 MHz (Cortex Technology, Denmark). The study was performed on 8 subjects, all aged 35–63 years, with clinical diagnosis of psoriasis and was approved by the Ethical Committee of the University of Medicine and Pharmacy “Iuliu Hatieganu” Cluj-Napoca, Romania, in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). After obtaining informed consent, each patient was treated with AgNPs cream and 1% hydrocortisone cream, respectively. 50 mg cream (containing 5 ␮g AgNPs)/cm2 skin were applied. The dose was selected on the basis of previous observations from a 14-day dermal toxicity test (unpublished data). All subjects were submitted to an ultrasonographic evaluation, the images being analyzed using Dermavision software. Data were acquired before and 2 weeks after the topically applied treatment (twice a day). 2.6. Statistical analysis The obtained data were analyzed using GraphPad Prism Software, version 5.0 (San Diego, CA). In order to make a comparison of multiple groups’ in the in vivo studies and in vitro cytotoxicity tests, one-way ANOVA with Tukey post test was used. All values in text and in figures are expressed as mean ± standard deviation, with a limit of statistical significance of p < 0.05. The Student’s t-test with n-2 freedom degrees was used for the correlation of the clinical and ultrasonographic aspects of the psoriatic lesions and computation of p-values was used. 3. Results and discussion The European black elderberry fruits extract is an important source of antioxidant compounds, among which polyphenols and especially anthocyanins play an important role. The water-soluble compounds from the aqueous extract of elderberries were found to be responsible for the reduction of the silver ions and the efficient stabilization of synthesized nanoparticles. The present study investigated the anti-inflammatory activity of the phytosynthetized AgNPs on different experimental models: HaCaT cells exposed to UVB radiation, carrageenan-induced paw edema in rats and psoriasis lesions in humans. 3.1. Characterization of nanomaterials Among the analytical techniques used to characterize the nanoparticles, UV–vis spectroscopy plays a significant role to authenticate the formation and stability of AgNPs in solutions. It is well known that the size of AgNPs influences their brown color, because of the excitation of surface plasmon resonance (SPR) of the nanoparticles [32]. Fig. 1a presents the UV–vis absorption spectrum of AgNPs. Distinctive peak may be observed at 426 nm with high absorbance which is characteristic for AgNPs. The stability of the AgNPs suspension was determined periodically by UV–vis spectroscopy. The solution was stable for 1 month, after which a significant shift was observed. The initial peak of 426 nm was red shifted to 448 nm after 40 days, showing the increase of the particles size (Fig. 1b).


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Fig. 1. (a) and (b). UV–vis spectra. (c) TEM image. (d) Histogram of biosynthesized AgNPs. (e) FTIR spectra of European black elderberry fruit extract and AgNPs. (f) Zeta potential of AgNPs.

Fig. 1c presents the TEM morphologies of the green synthesized nanoparticles. The TEM results revealed the obtaining of AgNPs with sizes varying within the range 20–80 nm, having almost spherical shapes. The obtained nanoparticles appeared to be collected in the form of a cluster. The corresponding size distribution is represented by a histogram of the AgNPs (Fig. 1d). The TEM images were in good agreement with the results obtained by UV–vis spectroscopy. The crude fruit extract and the purified AgNPs were analyzed by FTIR spectroscopy, in order to identify the biomolecules present in the extract liable for the reduction of the silver ions. Various modes of vibrations were identified and assigned to various functional groups. The strong stretching vibration of OH functional group can be observed at 3380 cm−1 for the crude extract and a frequency shift to 3428 cm−1 was observed in the case of AgNPs, indicating a cleavage of some H bondings (Fig. 1e). The band at 2936 cm−1 , due to the presence of C H stretching, was shifted to the lower

frequency (2921 cm−1 and 2857 cm−1 ) in AgNPs when compared to the extract. Vibrations at 1731 cm−1 were attributed to the C O group from the quinoidal form of polyphenols. After the reduction of Ag+ –Ag0 with natural extracts, the peak shifted to 1707 cm and markedly decreased in intensity, probably owing to the adsorption of natural polyphenols of the fruit extract on the surface of the AgNPs. To confirm the crystalline structure of synthesized AgNPs, the XRD spectrum was recorded. The X-ray diffraction studies pointed out the four characteristic Bragg’s XRD peaks at 2◦ values of about 38.2◦ , 44.3◦ , 62.2◦ and 77.9◦ corresponding to the crystallographic planes of faced centered cubic silver: {1 1 1}, {2 0 0}, {2 2 0} and {3 1 1}. The measurement of Zeta potential showed a sharp peak at −20.9 mV (Fig. 1f) indicating that the surface of the nanoparticle was negatively charged and giving further evidence that the silver nanoparticles were capped by polyphenols from the fruit extract.




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Fig. 2. Microscopic aspect of HaCaT cells: (a) non-treated, healthy cells; (b) HaCaT cells treated with AgNP. (c) and (d) survival curves for HaCaT cells treated with European black elderberry fruit extracts – nonlinear regression and four-parameter sigmoidal curve fit, each point representing mean ± SEM in three separate measurements; Dosedependent decrease of keratinocytes’ viability treated with AgNPs (g). * p < 0.05; ** p < 0.01; *** p < 0.001.

The electrostatic repulsive forces between the negatively charged nanoparticles may prevent the aggregation of the AgNPs, which might be responsible for their long term stability [33]. This property is also known to be involved in the surface interaction and cellular uptake of AgNPs [34]. The analysis of nanoparticles by means of inductively coupled plasma mass spectrometry resulted in a corresponding molar concentration of AgNPs = 4.10 nM.

3.2. In vitro studies 3.2.1. Effects on cell morphology and ultrastructure The uptake of nanoparticles by the cells is essential for the assessment of their effects at cellular level. It has been demonstrated that the most important characteristics for evaluating the cellular effects of nanoparticles are their size and shape. When administered in the same mass dose, small particles, having larger surface area exert higher toxicity [35]. AgNPs sizes were in the range of 20–80 nm as measured by TEM and had spherical shape, thus they can be considered middle to large nanoparticles. Chitrani et al., 2006 [36] demonstrated in their study that cellular uptake was the maximum for 50 nm gold nanoparticles and that spherical shape was an advantage. The uptake of nanoparticles can alter cellular function, but is likely that they have no negative effects. Nanoparticles interaction with the cellular surface can also have toxic effect on the cells, even if they are not internalized [37]. AgNPs are up taken by the cells by active mechanisms e.g. endocytosis or by passive mechanisms e.g. by diffusion. It is important to assess the dose–effect relationship, in order to define a safe range of AgNPs for the intended application having in mind that higher concentrations can have serious adverse effects [38,39].

Recent studies have pointed out that functionalization of AgNPs with compounds from fruits or plants reduced their cytotoxic effects. Thus, Moulton et al. (2010) [40] showed that epicatechinsynthesized silver nanoparticles had no toxic effects on HaCaT cells and induced no changes in cellular morphology. Silver nanoparticles synthesized using Albizia adianthifolia leaf caused no cytotoxicity on normal healthy human peripheral lymphocytes, the cell viability being higher than 100% [41]. In our study, HaCaT cells, after 24 h incubation with silver nanoparticles, were observed under a Zeiss AxioObserver D1 microscope equipped with a cooled AxioCamNR camera using AxioVision software. The morphologic changes in HaCaT keratinocytes as an effect of the exposure to increasing concentrations of AgNPs consisted in signs of significant cell alterations, the cells becoming round losing their characteristic epithelial cell shape and presenting intense vacuolization of the cytoplasm compared with control cells. No signs of massive cell death (floating cells) were present (Fig. 2a and b). TEM examination of the cells was useful in offering further data concerning the accumulation of nanoparticles in the keratinocytes. While control, non-treated cells were free of AgNPs (Fig. 3a and b), the AgNPs were found in high number in the treated cells. TEM revealed that the internalization of AgNPs in the HaCaT cells was through endocytosis (Fig. 3c–e). Thus, the AgNPs were observed in endocytosis vesicles, both at the periphery of the cells (Fig. 3c and d), and deep inside the cytoplasm (Fig. 3f). These vesicles had particular shapes and sizes, different as compared to the cell organelles. Moreover, a high number of electron dense vesicles (most likely lysosomes) were identified in many cells treated with AgNPs (Fig. 3c–f). In some of these vesicles the nanoparticles were identified as well (Fig. 3d and e). These observations were consistent with those published by Gliga et al. [42]. In order to elucidate the cellular and molecular mechanisms through the AgNPs influence the keratinocytes ultrastructure, a precise identification of




Fig. 3. TEM images of non-contrasted HaCaT cells: (a) and (b) Normal ultrastructural aspect of non-treated, healthy cells; (c)–(f). HaCaT cells treated with AgNP. Endocytosis vesicles containing AgNPs in relative high amounts are visible in the peripheral cytoplasm of cells (c)–(e) and also deep inside the cell (f). Electron dense vesicles (EDV – most likely lysosomes) are present in high number in many cells (c)–(f) and nanoparticles are visible in some of them (d) and (e).

these dense vesicles requires more experimental work to be done, including the use of fluorophores to establish whether the AgNPs stimulate indeed the proliferation of lysosomes, or the immunogold method to identify the vesicles containing the AgNPs as lysosomes. 3.2.2. Effects on cell viability After 24 h incubation with increasing concentrations of European black elderberry fruit extract, the viability of HaCaT keratinocytes decreased dose-dependently (Fig. 2c). The dose which reduced viability with 50% (inhibitory concentration 50IC50) was 79.4 ␮g/ml. HaCaT cells treated with AgNPs also illustrated a dose-dependent reduction of cell viability, which was significant starting with 23.7 ␮g/ml (p < 0.01) (Fig. 2d). 3.2.3. Effects on inflammation The interaction between UVB exposure, cytokines production and treatment with AgNPs of HaCaT cells was interesting and suggested their dual effect on cytokines secretion. The treatment of HaCaT cells with AgNPs led to an increased concentration of IL-1␣ in the culture media after 24 h incubation in comparison with control cells. After 48 h incubation, the situation was reversed, IL1␣ concentration being lower in the treated cells compared to the

non-treated counterparts (Fig. 4a). IL-1␣ is a chromatin – associated protein which is constitutively expressed in keratinocytes. Basal IL-1␣ is relatively high in keratinocytes, consistent with its role in proliferation [43]. Its release occurs whenever the cell is destroyed by necrosis or as a result of the action of several agents (LPS, lipopolysaccharide; UVB, etc.) which induce inflammation [44]. The first contact of HaCaT cells with AgNPs stimulated the release of IL-1␣ in the cell culture media. At 48 h, the IL-1␣ concentration was lower than in the supernatant of control cells, indicating that no further release occurred, probably as a result of the inhibition of its own production by IL-1␣, as it is known to have a dual function: it activates the transcriptional mechanism leading to the progression of inflammation and also acts as a signal transmitter mediated by receptors. IL-1␣ regulates its own production in a paracrine way but also the secretion of other pro-inflammatory cytokines: IL-6, IL-8, TNF-␣ [45]. The fruit extract had no effect on the release of IL-1␣, the values being the same as in control cells. When the keratinocytes were exposed to UVB, after 24 h, IL-1␣ levels were higher in the irradiated cells in contrast to the non-irradiated ones, both at 24 and 48 h. The pretreatment with AgNPs led to an additional increase (p < 0.001, two-way ANOVA, Bonferroni posttests), probably as an effect of cellular death by necrosis as a




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Fig. 4. (a) and (b) IL-1␣ concentration in the culture media of HaCaT cells 24 h after treatment with AgNPs and extracts and 24 h after treatment with AgNPs and extracts followed by exposure to UVB. c, d. IL-1␣ concentration 48 h after treatment with AgNPs and extracts and 48 h after irradiation with UVB.

result of the simultaneous action of UVB and AgNPs (Fig. 4b). At 48 h after exposure to UVB, significantly higher amounts of IL-1␣ were measured in the control culture media (exposed only to UVB), whereas the pretreatment with AgNPs reduced the concentrations of IL-1␣ versus the no treated irradiated cells (Fig. 4c and d). IL-6 is a cytokine that activates Jak-Stat signaling pathways, having both pro- and anti-inflammatory activity. Its expression is usually elevated in inflammatory diseases and is induced by IL-1␣ [46]. In our study, exposure to UVB led to a highly significant increase of the IL-6 levels at 24 h (p < 0.01) and 48 h (p < 0.001) after treatment as compared to the non-irradiated cells which is consistent with the results of Kirnbauer et al. (1991) [47] who found increased levels of IL-6 in the supernatants of cells exposed to UVB (100 J/m2 ). Incubation with AgNPs led to a significant reduction of IL-6 in the supernatants of HaCaT cells, while the crude extract had no effect on IL-6 secretion (Fig. 5a and b). When the AgNPs treated cells were irradiated, IL-6 concentrations were significantly lower than the irradiated and no treated cells, both at 24 and 48 h. This effect could be the result of an inhibitory effect of the nanoparticles as well as of the extract on the release of IL-6, as a result showing an anti-inflammatory effect (Fig. 5c and d). Nanoparticles produced for different purposes, due to their characteristics (size, shape, etc.) are able to enter human cells where they can produce several effects. Our findings suggest that AgNPs toxicity on human keratinocytes is dose-dependent, concentrations below 23.7 ␮g/ml producing no significant reduction of cell viability. Dose–effect relationship is important, in order to find the safe range of AgNPs concentrations for the intended application. When AgNPs came in contact with HaCaT cells they also led to the modulation of the release of IL-1␣ and IL-6. Inflammation is a physiological response

to various insults and acute inflammation represents an important defense mechanism of the body. 3.3. In vivo studies 3.3.1. Evaluation of paw edema by pletismometry Carrageenan-induced edema in the hind foot pad was used as a model to evaluate the temporal relationships between edema formation and the release of cytokines. Paw injection of carrageenan produced a significant increase in local swelling, with a maximum at 2 h after administration, the edema persisting up to 48 h after treatment (Fig. 6). The extract obtained from the fruits of European black elderberry exerted no significant anti-inflammatory effect at the administered dose (p > 0.05). AgNPs inhibited the inflammatory edema rate which was 12.61% at 2 h after the injection of carrageenan compared with control group. 3.3.2. Anti-inflammatory effects To identify the anti-inflammatory effect of AgNPs administration on paw edema model, the level of pro inflammatory cytokines, IL-1␣, IL-1␤ and IL-6 was measured, using a multiplex cytokine kit. Subplantar administration of carrageenan induced the secretion of pro-inflammatory cytokines reaching a maximum at 2 h after injection. In the paw tissue homogenates, the concentrations of these cytokines decreased to a minimum, early after the induction of inflammation, both with AgNPs as well as with the natural extract. Functionalization of AgNPs with polyphenols from European black elderberry fruit extract maintained these effects up to 48 h after carrageenan injection. Therefore, pretreatment with AgNPs significantly decreased IL-1␣ levels, both at 2 h after initiation of inflammation (87% inhibition; p < 0.01) as well as




Fig. 5. (a) and (d) IL-6 concentration in the supernatants of HaCaT cells 24 h after treatment with AgNPs and extracts and 24 h after irradiation with UVB and also 48 h after treatments without UVB and with UVB.

at 48 h (49% inhibition; p < 0.05) in comparison with the control group (Fig. 7a). The administration of European black elderberry fruits extract inhibited IL-1␣ secretion (57%) but not to statistically significant levels. A similar pattern was noticed in the secretion of IL-1␤, especially at 2 h after induction of inflammation (Fig. 7b). Pretreatments with fruit extract and AgNPs reduced IL-1␤ levels at 2 h after carrageenan injection in contrast to the control group (SN: 81%; p < 0.05; AgNPs: 92%; p < 0.01) and the difference gradually

decreased at 24 h and 48 h. In the treatment with fruit extract and AgNPs, IL-6 secretion was inhibited at 2 h after induction of inflammation (Fig. 7c). The effects were much lower than those of Indomethacine (p < 0.05). Consequently, European black elderberry fruit extract reduced IL-6 protein secretion by 74% (p < 0.05) and AgNPs by 83% (p < 0.01) in comparison with the group treated with saline solution. The effect was maintained up to 48 h of administration of AgNPs (35% inhibition).

Fig. 6. Paw edema evaluated before and after 2 h, 24 h and 48 h of carrageenan injection in Wistar rats pretreated with nanoparticles and natural extracts. AgNPs, SN extract, saline solution respectively Indomethacin were administered orally, 4 days consecutive days, prior to injection of carrageenan. The anti-inflammatory activity was evaluated by plethysmometry as described in “Methods”. Values are means ± SD. Statistical analysis was done by a one-way ANOVA, with Tukey’s multiple comparisons posttest.




9

Fig. 7. Pro-inflammatory cytokines levels in soft plantar tissue at 2 h, 24 h and 48 h after carrageenan injection in animals pretreated with AgNPs and natural extract. AgNPs and natural extracts were administered orally 4 consecutive days, prior to injection of carrageenan. Indomethacin was used as a positive control and 0.9% NaCl solution as a negative control. Values are means ± SD. Statistical analysis was done by a one-way ANOVA, with Tukey’s multiple comparisons posttest (*p < 0.05; **p < 0.001).

3.3.3. Assessment of anti-inflammatory effect by ultrasonography Psoriasis vulgaris is a chronic inflammatory disease characterized by well-defined erythematous plaques disseminated at cutaneous level, covered with white scales. In psoriasis, keratinocytes synthesize various pro-inflammatory cytokines, chemokines and growth factors. Subsequently, these affect Langerhans cells, lymphocytes, vascular endothelial cells and keratinocytes themselves to regulate immune responses and inflammatory reactions [48,49] and to stimulate the proliferation of keratinocytes. Despite synthetic AgNPs are being widely applied, there are limited data regarding the effects of AgNPs functionalized with natural extracts in human. There are few natural (chitosan) and synthetic polymers (aliphatic polyesters, polyacrylates) studied as targeted delivery systems for local treatment of psoriasis vulgaris [50]. These nanoparticles-based topical delivery formulations may be a promising therapeutic option in psoriasis, combining the advantages of both nanosized drug carriers and topical therapy. The anti-inflammatory effect of AgNPs in the treatment of the skin lesions that appear in psoriasis was investigated, by measuring the skin thickness of the patients, using a non-invasive ultrasonographic technique. The ultrasound assessment of skin lesions allowed the acquirement of cross-sectional images up to a depth of 2.5 cm. The histograms were analyzed with Dermavision software

that allowed measuring the number of pixels with different echogenicity, before and after therapy. The low echogenicity pixels (LEP) quantify the inflammatory process (lesion thickness) which decreased after therapy (see Fig. 8). After treatment, an improvement of skin lesions (reduced erythema, reduced induration) due to a decrease of the inflammatory process was clearly obvious. This was quantified by the hypoechogenic sub-epidermal band. The anti-inflammatory effect of the AgNPs cream was assessed on 8 patients. The results indicated a stronger effect of the AgNPs cream (50.78% decrease of skin thickness) as compared to hydrocortisone (42.21%, p < 0.05). Ultrasound characteristics of psoriasis plaques treated with AgNPs cream, confirmed the anti-inflammatory properties of the studied nanomaterials and the superiority of AgNPs compared to hydrocortisone cream. In all the experimental models, the inflammation is a common pathogenetic mechanism. The higher and more persistent antiinflammatory activity of AgNPs in comparison with fruits extract might be due to an enhanced permeability and retention effect of the AgNPs in the edema region and also to AgNPs uptake by the lymphatic system from the inflammation area. Although the AgNPs effects on inflammation are well documented, the mechanisms by which the nanoparticles, especially those functionalized




Fig. 8. (a) Ultrasound images: cutaneous histogram: normal aspect. (b) Psoriasis vulgaris: clinical aspect and histogram before treatment. (c) Psoriasis vulgaris: histogram after treatment with AgNPs.

with natural extracts, exert their bioactivity are not elucidated yet. Generally, the inflammatory response following carrageenan injection or UVB exposure involves mechanisms associated with prostaglandin production, COX-2 up-regulation and the formation of reactive nitrogen and oxygen species as well as cytokines and other inflammatory mediators. In these processes various cells are involved such as keratinocytes, Langerhans cells, neutrophils, vascular endothelial cells and lymphocytes. It has been demonstrated in vitro that UVB induces the secretion of IL-1␣ [51] while the expression of IL-1␣ and IL-1␤ genes in the keratinocytes is also enhanced [44]. IL-1␣ is a potent inducer of IL-6 production, and the administration of IL-1␣ antibodies led to the inhibition of IL-6 secretion after UV exposure. In vivo, the dynamics of cytokines secretion on a carrageenaninduced acute edema model differs from the results obtained in vitro. That is why the pre-administration of both AgNPs reduced the level of IL-1␣, IL-1␤ and IL-6 in the paw tissues, early after the induction of inflammation. The anti-inflammatory effects were maintained up to 48 h after treatment with AgNPs. These results suggested that AgNPs facilitated local accumulation and

persistence of natural extracts in the area of inflammation and thus bringing about long-term protective effects. 4. Conclusions European black elderberry fruits extract was used as reducing agent for the green synthesis of stable AgNPs. Their size (ranging between 20 and 80 nm) and shape (almost spherical) were confirmed by analytical characterization techniques. The synthesized nanoparticles presented a promising antiinflammatory effect, investigated both in vitro and in vivo. In vitro, the anti-inflammatory effect was demonstrated by the decrease of cytokines production and by maintaining their low level after UVB irradiation. In vivo, the pre-administration of AgNPs decreased the level of cytokines in the paw tissues and also presented long-term protective effect. The local treatment of psoriasis vulgaris skin lesions confirmed the good anti-inflammatory effect of AgNPs, which proved to be even better than that of hydrocortisone.




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