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Artificial Cells, Nanomedicine, and Biotechnology
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Antimicrobial properties of rosin acids-loaded
nanoparticles against antibiotic-sensitive and
antibiotic-resistant foodborne pathogens
Elisa Santovito, José das Neves, Donato Greco, Vito D’Ascanio, Bruno
Sarmento, Antonio Francesco Logrieco & Giuseppina Avantaggiato
To cite this article: Elisa Santovito, José das Neves, Donato Greco, Vito D’Ascanio, Bruno Sarmento, Antonio Francesco Logrieco & Giuseppina Avantaggiato (2018) Antimicrobial properties of rosin acids-loaded nanoparticles against antibiotic-sensitive and antibiotic-resistant foodborne pathogens, Artificial Cells, Nanomedicine, and Biotechnology, 46:sup3, S414-S422, DOI: 10.1080/21691401.2018.1496924
To link to this article: https://doi.org/10.1080/21691401.2018.1496924
Published online: 07 Dec 2018.
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Antimicrobial properties of rosin acids-loaded nanoparticles against
antibiotic-sensitive and antibiotic-resistant foodborne pathogens
Elisa Santovitoa , Jose das Nevesb,c,d, Donato Grecoa, Vito D’Ascanioa, Bruno Sarmentob,c,d , Antonio Francesco Logriecoaand Giuseppina Avantaggiatoa
a
Institute of Sciences of Food Production, National Research Council (ISPA-CNR), Bari, Italy;bi3S– Instituto de Investigac¸~ao e Inovac¸~ao em Saude, Universidade do Porto, Porto, Portugal;cINEB– Instituto de Engenharia Biomedica, Universidade do Porto, Porto, Portugal;dInstituto de Investigac¸~ao e Formac¸~ao Avanc¸ada em Ci^encias e Tecnologias da Saude, CESPU, Gandra, Portugal
ABSTRACT
Rosin acids (RA) from coniferous trees are used in folk medicine for healing various skin infections. Despite the antimicrobial potential of RA, their poor solubility in aqueous media may limit their use. In this work RA-loaded polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticles (RA-NPs) with enhanced antimicrobial properties against foodborne bacterial pathogens were produced. RA-NPs were prepared by solvent displacement technique and characterized for relevant colloidal features by dynamic light scattering, laser Doppler anemometry and transmission electron microscopy. Association of RA to NPs occurred with high yields (86% w/w). RA and RA-NPs (130 nm) were strongly active against antibiotic-sensitive Gramþ pathogens, i.e. Clostridium perfringens, Listeria monocytogenes and antibiotic-resistant Staphylococcus aureus. However, both failed in inhibiting the growth of Gram– pathogens (Campylobacter jejuni, Campylobacter coli, Escherichia coli and Salmonella enterica). Association to NPs enhanced the antimicrobial activity of RA. MIC, IC50, IC90, and MBC values of RA-NPs were ten-times lower than RA. RA-NPs did not change the intrinsic toxicity potential of RA. This is the first study on the enhancement of the antimicrobial activity of RA when associated to nanocarriers. This approach may be an effective strategy to produce aqueous-based RA solutions with enhanced antimicrobial activity against antibiotic-sensitive and antibiotic-resistant Gramþ pathogens.
ARTICLE HISTORY Received 17 May 2018 Revised 27 June 2018 Accepted 29 June 2018 KEYWORDS Nanoparticles; antimicro-bials; rosinic acids; pathogenic bacteria; nanocarriers
Introduction
Coniferous trees secrete resin at the sites of mechanical injury to prevent the invasion of pathogenic bacteria and fungi, and to deter herbivorous animals. Rosin acids (RA) are mainly extracted from the resin of Pinus species. The purified resin (rosin) contains hydrophobic diterpene carboxylic acids, mainly abietic, dehydroabietic, neoabietic, isopimaric, levopi-maric and palustric acids [1]. A number of in vitro tests dem-onstrated their efficacy as antimicrobials against a broad spectrum of microbes [2–4]. In clinical trials, rosin-based sal-ves have been proven to enhance the healing of skin infec-tions associated with wounds and ulcers [5,6]. Although the antimicrobial mechanism of action of RA have not been com-pletely elucidated, whereas transmission and scanning elec-tron microscopy and elecelec-tron physiology studies showed that the exposure of Staphylococcus aureus to rosin affected wall thickness, cell aggregation, fatty acids structure and mem-brane potential [7]. As a result, the membrane loses its integ-rity leading to structural and functional disruptions that affect the energy metabolism and cell viability [7,8].
Despite the potential of RA as antimicrobials, intrinsic hydrophobicity makes their use as modern aqueous-based antiseptics difficult [9–11]. Furthermore, when incorporated in
high doses, RA are known to exert topical toxic effects [12]. Recent attempts to increase the solubility of rosinic acids in water have been conducted by preparing aqueous oil-in-water emulsions [11], with substantial antimicrobial effects against bacteria and yeasts, including methicillin-resistant S. aureus.
Recent in vivo studies in broiler chicken showed the effi-cacy of RA in reducing the symptoms of Clostridium perfrin-gens induced necrotic enteritis, thus suggesting that the oral intake of RA may be helpful in treating or preventing gastro-intestinal diseases [13,14]. However, their use may be cum-bersome since several issues may arise, namely problems with palatability (if considering oral intake), stability either in storage or at biological settings, or toxicity due to the use of surfactants and other excipients used in nenoemulsions [15].
Nanotechnology has become an increasingly important technology for food safety, especially to boost the antimicro-bial potential of natural compounds, and is being used in for-mulating different delivery systems to protect loaded compounds from intrinsic and extrinsic factors such as pH, water activity, enzymatic degradation, temperature, relative humidity and storage environment [16]. For the formulation of poorly water soluble compounds, nanoparticles (NPs) based on synthetic polymers present several advantageous
CONTACTElisa Santovito elisa.santovito@ispa.cnr.it Institute of Sciences of Food Production, National Research Council (ISPA-CNR), Bari, Via Amendola 122/O, 70125, Italy
Supplemental data for this article can be accessedhere. ß 2018 Informa UK Limited, trading as Taylor & Francis Group 2018, VOL. 46, NO. S3, S414–S422
features [17]. For example, when suitably engineered, these carriers may present high physical–chemical stability [18], including when exposed to different gastrointestinal microen-vironments, potential to overcome biological barriers such as mucus [19–21], or enhanced ability to interact with cells par-ticularly at the membrane level [22]. NPs can also allow sus-tained release of active payload content and, among other benefits, reduce cell toxicity by avoiding peaking drug con-centrations at the surrounding environment [23]. Thereof, the incorporation of RA into NPs could have a beneficial impact, not only in enhancing antimicrobial activity but also the per-formance in biological settings such as in the gastrointestinal tract. This work explores the association of RA to polymeric NPs as an innovative approach to potentially deliver these hydrophobic compounds in aqueous-based media and test their antimicrobial activity against antibiotic-sensitive and resistant foodborne pathogens.
Methods
Rosin acids composition
The purified RA from Pinus eliottii was kindly provided by NFT S.r.l. (Lodi, Italy). The composition of RA, as provided by the manufacturer, and the chemical structure of the main constituents are detailed in Figure 1. RA contained palustric, abietic and neoabietic acids as main components.
Preparation and characterization of nanoparticles
RA-NPs were prepared by solvent displacement technique (nanoprecipitation). Briefly, 16 mg of polyethylene glycol-poly(-lactic-co-glycolic acid) (PEG-PLGA, Mn 5:55 kDa; Sigma-Aldrich,
St. Louis, MO, USA), 4 mg of amine terminated PEG-PLGA (H2N-PEG-PLGA, Mw 5:12 kDa; PolySciTech, West Lafayette, IN,
USA) and 5 mg of RA were dissolved in 1 ml of acetone. The mixture was then slowly injected into 10 ml of ultra-pure water under magnetic stirring (400 rpm). After 2 h, NPs were washed twice with 10 ml of water before being concentrated to a final volume of 1 ml using Amicon Ultra-15 filter units (MWCO 100 kDa; Merck Millipore, Tullagreen, Ireland). NPs without the incorporation of RA (empty NPs) were also simi-larly produced.
NPs dispersed in 10 mM sodium chloride were character-ized for hydrodynamic diameter, polydispersity index (PdI) and zeta potential using a ZetaSizer Nano ZS (Malvern, Worcestershire, UK). Furthermore, morphology and size con-firmation of NPs was performed by transmission electron microscopy using a JEM 1400 microscope (JEOL, Tokyo, Japan). Mean diameter values were calculated from image analysis of 150 individual NPs selected randomly using the elliptical selection tool of ImageJ software (v. 1.51j8, NIH, Rockville, MD, USA) and fitting data to a Gaussian distribution with the aid of PrismVR v. 5.03 (GraphPad Software, La Jolla,
CA, USA). NP samples were diluted 10-fold with water, placed onto nickel grids and stained with lanthanum acetate before observation at an acceleration voltage of 80 kV. The quantity of RA incorporated into NPs and the association efficiency of the preparation method were assessed by spectrophotomet-ric analysis. First, NPs were dissolved in ethanol at a ratio of 1:10 (particle dispersion:ethanol) for 1 h at room temperature, with 20 s vortexing at full speed every 15 min. Then, the amount of RA was determined at a wavelength of 241 nm using a Varioskan Flash spectrophotometer (ThermoFisher Scientific, Milan, Italy). A calibration curve was obtained by diluting known quantities of pure RA in ethanol in the range
Figure 1. Composition of the purified rosin. (a) Relative quantity of rosin acids is shown as percentage (w/w) as provided by the manufacturer and determined by GC-MS. (b) Chemical structure of main rosinic acids.
of 0–0.1 g/L. Empty NPs were treated in a similar way and used as blanks.
Bacteria and culture conditions
Liquid or agarified (1.5% agar) brain heart infusion broth (BHI, Oxoid, Milan, Italy) was used for S. aureus, Escherichia coli, Salmonella enterica and Listeria monocytogenes. Defibrinated sheep blood (Biolife Italiana, Milan, Italy) was added at 5% (v/v) for Campylobacter coli and Campylobacter jejuni culturing. Thioglycollate medium (Biolife Italiana) was used for C. perfringens. Authenticated MRSA strains were obtained from ATCC Standards. None of the other strains and isolates were declared as antibiotic resistant by the providers (Table 1). Bacteria were grown at 37C in aerobic conditions (S. aureus, L. monocytogenes, E. coli, S. enterica), microaerobic conditions (C. coli and C.jejuni) using CampyGen packs (ThermoFisher Diagnostics), and anaerobic conditions (C. per-fringens) using AnaeroGen pack (ThermoFisher Diagnostics) in airtight jars.
Challenge tests for antimicrobial activity
RA (5000 mg/L in acetone), RA-NPs (containing 430 mg/L RA) and empty NPs (no RA) were assayed versus Gramþ and Gram– bacteria. The antimicrobial effect was determined by culturing the target pathogens in the presence of RA or RA-NPs suspension. Sterile 96-well microplates were used, and tests were performed in a final volume of 100lL per well. In all cases, the antimicrobial:culture ratio was 1:9 to avoid excess RA/solvent in the medium. Each test contained ca. 105 CFU/mL of bacteria in the relevant medium. Blanks were obtained by adding sterile distilled water, acetone or sterile broth instead of NPs, RA or culture suspension respectively. Growth controls were obtained by adding sterile distilled water or acetone in place of the NPs or RA. The growth of bacteria was monitored by analyzing the turbidity in each
well using a microtitre plate reader (Varioskan Flash, ThermoFisher Scientific) at 600 nm. Each test was performed in triplicate wells, and the analyses were repeated in three independent experiments in different days. After a 24 h chal-lenge, a reduction in the OD600nmvalues in test samples as a
function of time was used as a measure of the antimicrobial activity. The number of microbial CFU was verified by plate counting onto agarified mediums detailed in the bacteria and culture conditions section.
MIC determination
To determine the minimum inhibitory concentration (MIC) the microtitre broth dilution method [24] was used, with the workflow proposed by Wiegand et al. [25]. Antimicrobials were tested in two-fold dilution series in the range of 0.01–0.4% w/v (125–4000 mg/L) for RA, and 0.65–10% v/v for the suspension of RA-NPs (containing 30–430 mg/L of RA). A final concentration of ca. 5 105 CFU/mL per well was used for each tested bacterium. Growth controls and blanks were obtained as described above. Plates were incubated at 37C for 24 h in the required conditions. Growth was monitored by analyzing the turbidity in each well using a microtiter plate reader as described above. The cell viability was confirmed by resazurin staining [26]. The experimental MIC (MICe) is the
concentration of the higher dilution well in which the absence of bacterial growth occurred, with no visible turbid-ity and no significant change in the OD600nm value, and a
blue staining observed after resazurin addition. The minimum bactericidal concentration (MBC) was determined as the high-est dilution at which no growth occurred in media. MBC was confirmed by plate counting. Each test was replicated in three wells, while three independent experiments were repeated in different days.
Cytotoxicity assay
The toxicity of RA and RA-NPs to the Caco-2 human cell line (C2BBe1 clone; ATCC, Manassas, VA, USA) was determined by the thiazolyl blue tetrazolium bromide (MTT) metabolic activ-ity assay and the lactate dehydrogenase (LDH) release assay. The first assesses mitochondrial activity, while the second evaluates cell membrane integrity. In brief, Caco-2 cells at passages 70–72 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Lonza, Verviers, Belgium) supple-mented with 10% (v/v) fetal bovine serum (Merck Millipore, Burlington, MA, USA), 100 U/mL penicillin (Merck Millipore) and 100lg/mL streptomycin (Merck Millipore), at 37C, 5% CO2 and 95% relative humidity. Cells were seeded at a
density of 104 per well in 96-well plates and left overnight under culturing conditions before being incubated for 24 h with concentrations of RA or RA-NPs between 106and 101mg/mL (expressed as RA content) in supplemented DMEM (for MTT assay) or plain DMEM (for LDH assay). A min-imal amount of dimethyl sulfoxide (0.1% or less) was used to allow dispersing RA in media. In the case of the MTT assay, medium containing either RA or RA-NPs was then discarded, cells washed thrice with phosphate buffered saline (pH 7.4)
Table 1. List of bacterial strains and the acronyms used in the text.
Species Strain name Gram ± Aerobic ± Source Acronym
S. aureus MSSA LMG 16811 þ þ LGC MSSA
S. aureus MRSA 1063 þ þ ATCC MRSA
S. aureus MRSA HDE288 þ þ ATCC
S. aureus MRSA M10/0148 þ þ ATCC
S. aureus MRSA NYBK2464 þ þ ATCC
S. aureus MRSA HPV107 þ þ ATCC
S. aureus MRSA HFH30364 þ þ ATCC
S. aureus MRSA B8-31 þ þ ATCC
E. coli O157 AD490 – þ ADRIA EC
E. coli O157:H7 AD565 – þ ADRIA
E. coli ATCC 35401 – þ ATCC
L. monocytogenes AD612 þ þ ADRIA LM
L. monocytogenes AD273 þ þ ADRIA
S. enterica ATCC 13311 – þ ATCC SE
C. perfringens ATCC 13124 þ – ATCC CP
C. perfringens CP56 þ – UGhent
C. perfringens AD211b þ – ADRIA
C. perfringens AD1600 þ – ADRIA
C. jejuni LMG 8841 – – LGC CJ
C. coli LMG 6440 – – LGC CC
ATCC: American Type Culture Collection, Manassas, Virginia, USA; ADRIA: Adria Developpement, Quimper, France; LGC: LGC Standard, Milan, Italy; U-Ghent: Prof. F. Van Immerseel, University of Ghent, Ghent, Belgium.
and incubated with MTT (0.5 mg/mL) in supplemented DMEM. After 4 h incubation at culturing conditions, medium was discarded, newly formed formazan crystals dissolved with dimethyl sulfoxide and absorbance measured at 570 nm using a Synergy 2 Multi-Mode plate reader (BioTek, Winooski, VT, USA). Results were expressed as viability percentage by comparing with absorbance values for cells incubated for 24 h with medium alone. As for the LDH assay, a commer-cially available kit from Takara Bio (Shiga, Japan) was used according to the manufacturer’s instructions in order to determine the amount of LDH from cell supernatants after incubation with RA or RA-NPs. Results were expressed as cytotoxicity percentage by considering the values for cells incubated with medium only or Triton X-100 (2%, w/w) as 0% and 100% toxicity, respectively. Experiments were per-formed in triplicates for both MTT and LDH assays. Half-max-imal cytotoxic concentration (CC50) values were also
calculated by logistic regression of concentration vs. normal-ized response data.
Data processing and statistics
Data processing of antimicrobial activity was performed according to du Toit and Rautenbach [27,28] in order to obtain analytical dose–response data. The OD600nm values of
strains from the same species were pooled and averaged. These data were used to measure the percentage of growth as follows:
% growth ¼ 100 sample OD – blank OD OD of growth control – blank OD These data were plotted as function of RA or RA-NPs con-centration, and fitted using nonlinear regression method by the SigmaPlot 12 for Windows (Systat Software, San Jose, CA, USA). The sigmoidal curve obtained for each data set, with variable slope, was fitted using the following mathematical model:
y¼ min þ max – min 1þ x=
IC50
Hill slope
Where min is the y-value at the bottom plateau; max is the y-value at the top plateau; IC50is the x-value of response
halfway between minimum and maximum values; Hill slope is the Hill coefficient or slope factor (controls slope of curve). MIC and the IC90 were calculated from the x-values at the
intercept between the slope and the x-value of y = 10, respectively.
In order to estimate the content of RA in RA-NPs, drug loading (DL%) was calculated taking into account the per-centage of RA incorporation and the amount of all other components (PEG-PLGA and H2N-PEG-PLGA):
DL%¼ mass of RA
total mass of RA NPs 100
Statistical analysis was performed by SigmaPlot 12 Software. Results are presented as mean ± standard deviation among replicates unless otherwise mentioned. Growth data were analyzed by ANOVA and followed by All Pairwise
Multiple Comparison Procedures (Tukey’s test), with a .05 sig-nificance level.
Results
Production and characterization of the nanoparticles
RA-NPs were successfully produced by solvent displacement technique. Dynamic light scattering (DLS) measurements revealed that NPs featured mean hydrodynamic diameter of 131 ± 1 nm and PdI of .155±.002. RA-NPs were larger and less mono disperse when compared to empty NPs (93 ± 2 nm and .075±.009). These data indicate that incorporation of RA into NPs induced slight aggregation. TEM imaging (Figure 2) con-firmed the colloidal and relatively homogeneous size distribu-tion of RA-NPs. Still, mean diameter values determined from image analysis and Gaussian distribution fitting (66 ± 15 nm), were lower as compared to DLS measurements. Such differen-ces are typical when considering different measurement tech-niques [29] and, in the particular case of the present work, may reflect size overestimation by DLS due to some particle aggregation. Interestingly, similar diameter values for RA-NPs and empty NPs (64 ± 19 nm) as determined by TEM analysis and Gaussian distribution fitting further confirm the ability of RA to induce aggregation. ZP values for RA-NPs and empty NPs were near neutral (–1.3 ± 0.1 mV and –3.2 ± 0.1 mV, respectively, as determined by laser Doppler anemometry), thus indicating surface shielding with PEG chains. The pres-ence of roughly 20% of H2N-PEG-PLGA in the formulation of
NPs was not able to shift ZP considerably towards positive val-ues. The amount of RA into NPs was assessed by extraction with ethanol and subsequent spectrophotometric assay. Association efficiency was 86.0 ± 0.2% (w/w), corresponding to a total concentration of 4,300 ± 100 mg/L of the final suspen-sion of NPs. These values indicate high drug content of NPs, corresponding to an estimated18% (w/w) drug loading.
Antibacterial challenge tests
Taking into account the yield of the encapsulation reaction, the concentrations of RA assayed in this test were 5000 mg/L as RA, 430 mg/L as RA-NPs. As shown inFigure 3, the growth of all pathogens was not influenced by the presence of empty NPs (i.e. without RA). This result suggests that PEG-PLGA and H2N-PEG-PLGA alone and organized as NPs did not
interfere considerably with the bacterial growth. Also, no
Figure 2. TEM image analysis of NPs. (a) Representative TEM image of RA-NPs (scale bar¼100 lm). (b) Gaussian fitting (black line) of the size distribution of NPs (grey bars).
effect of acetone on the growth of bacteria was found. As shown inFigure 3andSupplementary Tables 1 and 2, after a 24 h challenge with the highest concentrations of RA (5000 mg/L) and RA-NPs (430 mg/L), marked antimicrobial activity towards Gramþ bacteria (i.e. S. aureus, C. perfringens and L. monocytogenes) was found. Even at these maximum concentrations, RA and RA-NPs failed in inhibiting the growth of Gram– bacteria (i.e. C. jejuni, C. coli, E. coli and S. enterica). Among Gramþ bacteria, RA and RA-NPs did not display dif-ferences between species and within species, although at concentration levels 10-fold higher for RA than for RA-NPs. When the growth kinetics of S. aureus LMG16811, C. perfrin-gens ATCC13124, and L. monocytogenes AD612 were analyzed (Figure 4(a–c), respectively), complete growth inhibition was observed in less than 8 h using RA-NPs. In the kinetics experiments we used RA at the dosage of 500 mg/mL, in order to compare the effect of RA when they are in the free form are loaded into NPs. At this dosage, free RA were not
effective in reducing the growth of the target pathogens. NPs did not affect the growth of the bacteria.
Determination of MIC, IC50, IC90 and MBC
The experimental MIC (MICe) values determined by the
micro-titre broth dilution assay were 2000 mg/L for RA and 215 mg/L for RA-NPs for all the MRSA and MSSA strains of S. aureus, L. monocytogenes and C. perfringens (Supplementary Table 1 and 2). When RA were used at the concentration of 500 mg/L, no reduction in the growth of the pathogens was observed. For these pathogens, 4000 and 430 mg/L were the MBC values determined for RA and RA-NPs, respectively (Figure 5).
The calculated MIC, IC50, and IC90 values for RA and
RA-NPs are listed in Table 2. For RA, MIC values ranged from 1485 ± 12 to 1711 ± 17 mg/L and differed significantly among species. IC50values ranged from 1163 ± 13 to 1615 ± 12 mg/L
with significant differences among species. IC90 values were
Figure 3.Challenge tests on pathogenic bacteria. Results represent mean ± standard deviation of nine OD600nmmeasures per group. The corresponding Log[CFU/ mL] are reported on secondary axis. Each strain was incubated for 24 h with empty NPs (NP0), no drug (CTRL), RA-NPs (containing 430 mg/L RA), or 5000 mg/L RA at the optimal growing temperature for each pathogen.
in the range 1482 ± 10 to 1692 ± 12 mg/L, with no significant difference between MRSA and CP. For RA-NPs, the calculated MIC values ranged from 154 ± 1 to 158 ± 1 mg/L, with signifi-cantly higher values for MRSA and MSSA than CP and LM. IC50 values were significantly different among species and
ranged from 133 ± 1 to 152 ± 1 mg/L. IC90 values were in the
range of 149 ± 1.1 to 157 ± 1 mg/L.
Cytotoxicity
The MTT and LDH assays were used to determine the in vitro toxic effects of RA-NPs and free RA on the human Caco-2 cell
line. These cells are considered as representative of human enterocytes and are widely used in different cell-based mod-els to assess different features of drugs and delivery systems intended for oral administration, namely their toxicity [19]. Results for cell viability/toxicity assays are presented in
Figure 6. The incorporation of RA into NPs did not seem to change the profile of RA thus suggesting that the proposed formulation did not enhance toxicity. Values of CC50 further
reinforce visual inspection of viability/toxicity profiles. In the case of the MTT assay, values of 31 mg/L (expressed in RA content) and 35 mg/L were calculated for RA-NPs and RA, respectively. As for the LDH assay, CC50 values were 43 mg/L
for RA-NPs and 28 mg/L for RA. Moreover, empty NPs did not induce noticeable change to Caco-2 cells within the consid-ered concentration range (CC50values above 100 mg/L), thus
suggesting that toxicity was mainly induced by the presence of RA, not particles or their components.
Discussion
Nanoemulsions, microemulsions, solid-lipid nanoparticles and liposomes are some of the currently used strategies to encapsulate plant bioactive compounds [16]
In this study we showed that the incorporation of RA into polymeric NPs may be a promising approach to deliver these hydrophobic compounds in aqueous media and to enhance their antimicrobial effect against foodborne pathogens. RA
Figure 4.Growth curves of S. aureus, C. perfringens and L. monocytogenes. Strains MSSA-LMG16811 (a), CP-ATCC13124 (b) and LM-AD612 (c) were grown with 10% RA-NPs (RA-NPs), 500 mg/L of free RA (RA) and empty NPs (NP0). The empty NPs and free RA groups were considered as control. Results are pre-sented as mean ± standard deviation (n¼ 3).
Figure 5. Dose–response curves from MIC experiments. The MIC, the IC50and the IC90values for RA (a) and RA-NPs (b) were calculated from the graphs. Asterisk indicates the MICevalue. Data are reported as mean and standard devi-ation (n¼ 3).
and RA-NPs displayed antimicrobial activity against Gramþ bacteria, including the antibiotics-resistant strains of S. aureus. These data are in accordance with previous studies that attributed the antimicrobial activity of RA to their lipidic nature that could lead to their accumulation in the plasma membrane, thus interfering with the energy metabolism of cells, and with higher affinity to Gramþ bacteria than Gram– bacteria [1,30–32].
Interestingly, the MIC, IC50, IC90, and MBC values of
RA-NPs were 10-fold lower than RA. Furthermore, when used at the same MIC dosage incorporated into NPs, free RA did not affect the growth of the pathogen, thus indicating that the loading of RA into NPs enhances the antimicrobial activity.
Although we did not look into the mechanisms behind the increased activity of RA once associated to NPs, it seems feasible to conjecture that such observation is related with enhanced binding of nanosystems to bacteria. In particular, the incorporation of positively-charged amine-terminated PEG (H2N-PEG-PLGA) is likely to have improved the
electrostatic interaction of NPs with negatively charged pepti-doglycans of the bacterial cell wall as previously shown [33–35]. In our hypothesis, this effect may contribute to attract NPs onto bacteria, increase the amount of RA released at the membrane level, and enhance the disruption of the bacterial membrane induced by RA [16]. As a consequence, damages induced into the cell wall may result in osmotic lysis of the cells [36]. Furthermore, the incorporation of RA into NPs may augment RA dispersability in aqueous media, thus increasing the effective amount of RA interacting with bacteria. Martins et al. [37] found a similar effect by using PLGA NPs for formulating poorly water-soluble violacein nanoantibiotics, with enhanced antimicrobial activity.
From a practical point of view, the increased antimicrobial activity observed for RA-NPs have important implications for their potential applications. The MIC values of RA-NPs are about 10 thousand-times lower than the quantity of RA used in traditional salves and creams [4,5], and more than t10-times lower than the inclusion level of RA used in in vivo studies with broiler chickens challenged with C. perfringens [13,14]. In this last study RA were found efficacious in increas-ing body weight, and in reducincreas-ing necrotic enteritis-associated sympthoms [13]. Overall, these studies suggest that dietary inclusion of RA-NPs has the potential to act as a perform-ance-enhancer and microbial modulator in farm animals.
The broad-range antimicrobial activity exerted by RA-NPs against Gramþ bacteria indicates that the mechanisms of action of RA-NPs are general and antiseptic-like, and distin-guishable from those typical antibiotics. Furthermore, RA-NPs have the potential to be efficiently used as an anti-MRSA drug. Upon incorporation into NPs, RA may be more effi-ciently delivered to target bacteria in aqueous environments and thus their antimicrobial activity gets enhanced. Finally, materials used for producing NPs are generally regarded as safe for medical use and should not be a cause of worries regarding toxicity [38]. In this perspective, the association of RA to NPs may constitute a viable and safe way to deliver RA orally. Indeed, the cytotoxicity tests performed with RA-NPs showed that the incorporation of RA into NPs did not change the toxicity of RA. However, toxic levels of RA and RA-NPs to Caco-2 cells were in close proximity to those required for antimicrobial activity of RA. These data are in accordance with previous studies which demonstrated that antibacterial activity of RA parallels cytotoxic activity, which suggests a similar membrane-associated mode of action [12].
To reduce cytotoxicity of RA-NPs, structural modification of RA can be undertaken with view of improving on their safety [11]. Furthermore, acute toxicity tests on mice would
Table 2. MIC, IC50and IC90values for RA and RA-NPs.
RA RA-NPs
MIC mg/L IC50mg/L IC90mg/L MIC mg/L IC50mg/L IC90mg/L MRSA 1485.0 ± 12.5a 1441.9 ± 13.3a 1476.3 ± 12.2a 157.7 ± 1.1b 151.9 ± 1.1a 156.5 ± 1.1a MSSA 1645.9 ± 15.6b 1248.5 ± 12.5b 1557.4 ± 10.5b 157.3 ± 1.0b 141.6 ± 1.1b 154.0 ± 1.0b LM 1711.2 ± 17.3c 1615.4 ± 12.5c 1691.6 ± 12.0c 153.8 ± 1.0c 133.1 ± 1.0c 149.3 ± 1.1c CP 1575.4 ± 12.2d 1163.8 ± 13.2d 1482.8 ± 10.4a 150.0 ± 1.0c 133.8 ± 1.0d 150.5 ± 1.1c Data are reported as average ± standard deviation among different strains for each species. Data were calculated by curve fitting inFigure 5. Different superscript letters indicate statistically sig-nificant differences among species for each parameter (in columns), according to ANOVA and Tukey’s post-hoc test, (p < .001).
Figure 6. Toxicity of RA-NPs, NP0 and RA to Caco-2 cells. (a) Cell viability of Caco-2 cells exposed to RA-NPs, empty NPs (NP0) or RA for 24 h as determined by the MTT assay. (b) Cytotoxicity effects of RA-NPs, empty NPs (NP0) or RA to Caco-2 cells after 24 h exposure as determined by the LDH assay. In both graphs, individual points represent mean viability/cytotoxicity values and verti-cal bars the standard deviations (n¼ 3), while lines stand for the logistic regres-sion of experimental points.
also shed a light on the toxicity of RA-NPs in vivo. To the best of our current knowledge, this is the first report on the antimicrobial activity of RA-NPs.
Acknowledgements
The authors thank Prof. Filip Van Immerseel for providing the C. perfrin-gens CP56 strains and ADRIA developpement for providing the strains indicated with the letters AD in this study. The authors also thank NFT S.r.l. for providing purified rosinic acids, and the i3S Scientific Platform Biointerfaces and Nanotechnology (BN) for assistance in DLS/LDA measurements.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Elisa Santovito http://orcid.org/0000-0001-8793-6351
Bruno Sarmento http://orcid.org/0000-0001-5763-7553
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