Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) is challenging modern antimicrobial therapy due to its high antimicrobial resistance. Nutraceuticals have gained a lot of interest and their incorporation into nanoparticles further improves their efficacy. This study aimed to evaluate the antibacterial activity of linalool-based lipid nanocapsules loaded with resveratrol (LIN-LNC-RES) as a synergistic strategy against MRSA. LIN-LNC-RES were prepared by the phase inversion temperature method and characterized for their colloidal properties, in vitro release, and stability. The antibacterial and antibiofilm activity against S. aureus and different MRSA clinical isolates were investigated. Furthermore, scanning electron microscopy (SEM) imaging for visualization of biofilm formation and bacterial membrane integrity as well as mechanistic investigation using quantitative real-time polymerase chain reaction (qRT-PCR) analysis were performed. LIN-LNCs-RES demonstrated favorable properties with a size of 35.19 ± 0.72 nm, PDI of 0.09 ± 0.02 and a zeta potential of -2.53 ± 0.07 mV with RES 98% EE. They showed a controlled release of RES over 24 h and were stable at 4 °C for 3 months. Compared to free drug, LIN-LNC-RES showed a 4-fold decrease in MIC values and 10-fold decrease in half maximal biofilm inhibitory concentration value. Biofilm eradication assay showed superiority of LIN-LNC-RES over RES against all isolates with disrupted bacterial membranes as revealed by SEM. Mechanistically, qRT-PCR showed that LIN-LNC-RES significantly reduced RNAIII gene expression as well as the expression of SaeRS two component system, potentially affecting quorum sensing and virulence factors expression. RES-loaded LIN-based nanosystem offers a great potential for combating MRSA infections, neutralizing its virulence activity hence, overcoming antimicrobial resistance.
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Introduction
Methicillin-resistant staphylococcus aureus(MRSA) is a global health-threatening pathogen responsible for more than 100,000 deaths in 2019 with a 64% mortality rate1. MRSA causes a wide range of infections such as sepsis, pneumonia, meningitis, endocarditis, skin, soft tissue and bone infections2. The multi-drug resistance (MDR) patterns of MRSA extended beyond penicillin to include a wide range of antimicrobials such as tetracyclines, aminoglycosides, fluoroquinolones and macrolides3. Currently, antibiotics such as vancomycin, linezolid, daptomycin, telithromycin are clinically indicated for the treatment of MRSA infections4. However, resistance against these antibiotics especially vancomycin, has emerged rendering treatment ineffective5. Biofilm formation is an intricate factor contributing to MRSA virulence resulting in prolonged and recurring infections6. The presence of the bacteria within the biofilm imparts a protective mechanism against antimicrobials leading to the emergence of MDR strains. Therefore, alternative approaches to conventional antibiotics are being investigated in an attempt to overcome MDR MRSA strains. These include antimicrobial peptides, phage therapy, phytochemicals, antibodies/vaccines and nanoparticles6.
Nanoparticles (NPs) offer a promising solution to overcome MDR MRSA owing to their small particle size (1–100 nm) and large surface area7. Several studies investigated the safety and efficacy of different categories of NPs (lipid-based, polymeric, peptide-based and metallic) as antibacterial delivery systems8,9,10,11. Lipid nanocapsules (LNCs) are considered a hybrid between liposomes and polymeric NPs consisting of an oily core surrounded by a lipophilic surfactant (lecithin) and a pegylated non-ionic surfactant rigid outer shell. LNCs are prepared by phase inversion temperature (PIT) method which is a low-energy, solvent-free method12. LNCs possess several advantages over other lipid-based nanocarriers such as their small size (25–100 nm) with a narrow size distribution, higher encapsulation efficiency, higher stability, longer circulation time, biocompatibility, biodegradability and ease of manufacturing12. LNCs have been widely used in various biomedical applications such as cancer, pulmonary, dermatological, cardiovascular, ocular and infectious diseases8,13,14. LNCs demonstrated high efficacy and biocompatibility as delivery systems for various antimicrobial compounds against MRSA as shown in several studies15,16.
Nutraceuticals are naturally occurring bioactive compounds possessing pleiotropic effects that have been used in the treatment of various condition including infectious diseases17. Among various classes of nutraceuticals, phytochemicals demonstrated antibacterial activity against various organisms especially MRSA18,19. Resveratrol (RES) is a polyphenolic compound that belongs to the stilbene family and is naturally found in peanuts, grapes, cranberries and blueberries20. RES has been widely used as an anti-inflammatory, anti-cancer, antioxidant and anti-ageing compound. RES also demonstrated antiviral, antifungal and antibacterial activity gram positive and gram negative bacteria including MRSA21,22,23,24. However, RES has some unfavorable physicochemical properties that limited its use these include its high hydrophobicity, low solubility, low bioavailability and chemical instability (photo-degradation/thermal degradation) in addition to its rapid and extensive metabolism and excretion24,25. Therefore, the development of delivery systems to improve RES physicochemical properties is crucial to enhance its therapeutic applicability. Different nanocarriers such as polymeric micelles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), liposomes, and lipid core nanocapsules have incorporated RES and were shown to enhance its therapeutic activities26,27,28. However, very few studies investigated the efficacy of RES-loaded LNCs in various applications. RES-loaded LNCs showed more pronounced effects as compared to free RES in bleomycin-induced acute and chronic lung disease29.
Essential oils are complex mixtures of volatile oils that can be extracted from aromatic plants and mainly consist of terpenes, 80% of which are monoterpenes30. Essential oils such as thymol, carvacrol, orange oil, eucalyptus oil, cinnamon, clove, and lavender possess antibacterial activity against a variety of gram-positive and gram-negative bacteria30,31. Linalool (LIN) is a monoterpene tertiary alcohol found in the essential oils of over 200 plants32. LIN has inherent anti-inflammatory, antiproliferative, anticancer, analgesic, neuroprotective, antihyperlipidemic and antimicrobial activity. LIN exhibited additive antibacterial activity against gram positive and gram negative bacteria when used in combination with other essential oils32. Moreover, anti-biofilm activity of LIN against E. coli, salmonella, and Campylobacter spp.has been established allowing its use in the food industry33. However, its high lipid solubility which results in low bioavailability limits its clinical use. Thus, the incorporation of LIN in lipid-based nanoparticles has been investigated in several studies and showed an enhancement in its pharmacokinetic profile and its therapeutic efficacy34,35,36. To date, incorporation of LIN into LNCs as an integral component of LNCs to develop an antibacterial nanosystem against MRSA has not been studied, yet.
Green nanotechnology has become a global demand to reduce environmental hazards through engineering eco-friendly nanosystems37. It is based on using simple, cost-effective, readily available materials without harmful chemicals thus preventing the production of toxic by-products. Green nanomedicine can be implemented through the utilization of preparation methods that apply the principles of green nanotechnology in addition to relying on naturally occurring bioactive compounds as raw materials37,38. The preparation of LNCs by PIT method and the incorporation of natural bioactive compounds such as LIN and RES align with the emerging trend of preparing eco-friendly nanosystems using green nanomedicine.
Therefore, the aim of the present study was to design and characterize eco-friendly, LIN-based LNCs loaded with RES to investigate their antimicrobial as well as anti-virulence properties against staphylococcus aureus strains, including MRSA isolates. A mechanistic investigation was also performed via the identification of specific genes targeted by RES-loaded LNCs using quantitative Real-time polymerase chain reaction (qRT-PCR).
Materials
Resveratrol (RES) was supplied by Ningbo Liwah Pharmaceutical Co., Ltd. (Zhejiang, China). Labrafac® (lipophile WL 1349, caprylic-capric acid triglycerides, European Pharmacopeia, IVth, 2002, was a kind gift from Gattefossé S.A., Saint-Priest, France). Kolliphor® HS 15 (Solutol® HS 15), a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate, European Pharmacopeia, IVth, 2002 was purchased from BASF (Ludwigshafen, Germany). Lipoid® S100 (soybean lecithin, 69% of phosphatidylcholine was a kind gift from Lipoïd, GMBH, Ludwigshafen, Germany. Linalool, 97% ((±) 3,7-Dimethyl-3-hydroxy-1,6-octadiene) was obtained from Sigma –Aldrich (Saint-Quentin Fallavier, France). All other chemicals and solvents were of analytical grade.
Methods
Preparation of lipid nanocapsules
Blank Lipid nanocapsules (LNCs) were prepared by the phase inversion temperature (PIT) and dilution method as previously described12. Kolliphor® HS 15, Labrafac®, and deionized water were mixed in a weight ratio of (1:1:3) by magnetic stirring. NaCl (0.44%w/v) and Lipoid® (0.75% w/v) were further added to the oil/water/Surfactant mixture. The formulation was subjected to 3 heating/cooling cycles between 65 and 85 °C. In the last cycle, quenching was induced at the PIT (82 °C) by a 3.5-fold dilution using cold deionized water (0–2 °C). This was followed by slow magnetic stirring for 5 min, filtration through a 0.45 μm syringe millipore filter, and storage at 4 °C until further characterization.
For Linalool-based LNCs (LIN-LNCs), and RES -loaded LNC (LIN-LNC-RES); Linalool (7% w/v) and RES (0.1–0.3 g %) were simply mixed with all other ingredients from the beginning and proceeded as before with Blank LNCs but at lower temperature ranges. For (LIN-LNC-RES), the formula was protected from light throughout the process.
Physicochemical characterization of LNCs
Colloidal properties
The z-average particle size and polydispersity index (PDI) were measured by dynamic light scattering (DLS) using Malvern Zetasizer® at a fixed angle (173°) at 25 °C using a 4mW He-Ne laser at 633 nm (Zetasizer® Nano ZS series DTS 1060, Malvern Instruments S.A, Worcestershire, UK). Zeta potential measurements were determined at 25 °C in water (dielectric constant 79, refractive index 1.32, viscosity 0.88 cP) using a cell voltage of 150 V and 5 mA current. Prior to measurements, samples were diluted 1:20 v/v with filtered deionized water. Measurements were performed in triplicate.
Morphology
Blank LNCs, LIN-LNCs and LIN-LNC-RES (2 mg/ml) were examined by transmission electron microscopy (TEM, JEM-100 CX, JEOL, Japan). LNC dispersions were diluted (1:60 v/v) with deionized water followed by spraying onto copper grids and staining with 2% w/v uranyl acetate solution. Samples were then allowed to dry under ambient conditions and shots were taken at x 50 K at 80 kV.
Entrapment efficiency and drug content
The percent entrapment efficiency (EE %) was determined by ultrafiltration/ultracentrifugation method39. LNCs dispersion was added to an ultracentrifugal concentrator (Sartorius™ Vivaspin6™, MWCO 100,000) and centrifuged for 30 min at 3663 × g at 4 °C. The amount of un-entrapped RES separated in the filtrate was determined spectrophotometrically at λmax = 303 nm against blank formulation as a reference. RES concentration was calculated using calibration curve standards (linearity range 0.8–5 µg/ml) according to the following equation:
Drug content was calculated by dissolving LIN-LNC-RES in tetrahydrofuran (THF) and methanol (1:18) followed by sonication for 5 min. Then, the mixture was further diluted 25 folds followed by sonication. The amount of the drug in the concentrate was determined spectrophotometrically at λmax 303 nm.
Differential scanning calorimetry (DSC)
The thermal behavior of the free RES, LIN, physical mixture 1 (without the drug), physical mixture 2 (with RES), Blank LNC, LIN-LNC and LIN-LNCs-RES were assessed using a differential scanning calorimeter (DSC) (PerkinElmer Inc, USA). First LNC dispersions were dried overnight in a desiccator after mixing with Aerosil®at a ratio of 4:1 w/w40. Then, 5 mg of powdered samples was placed in sealed aluminum pan and a temperature ranging from 25 to 300 °C was scanned at a heating rate of 10 °C/minute. Inert atmosphere was maintained by purging nitrogen at a flow rate of 20 mL/min.
Fourier-transform infrared spectroscopy (FT-IR)
The interaction between RES and LNCs components was investigated using FTIR (Model- Frontier, Perkin Elmer, Waltham, USA)41. A small amount of aerosil-dried preparations were mixed with potassium bromide and then pressed into pellets to be analyzed. Spectral analysis was performed at a wavelength range between 4000 and 500 cm−1 at a resolution of 4 cm−1 with an average of 100 scans.
In-vitro drug release study
RES release from LNCs was determined by dialysis method42. Briefly, presoaked dialysis bags (Visking®36/32, 24 mm, MWCO 12,000–14,000, Serva, USA) were filled with 0.5 ml LIN-LNC-RES or RES solution in 70% ethanol (2 mg/ml). To maintain sink conditions, 50 ml of phosphate buffer saline (PBS), pH 7.4 was used as the release media42. Samples were placed in a thermostatically controlled shaking water bath at 37 °C at 100 rpm. At specified time intervals (0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h), 1 ml of the release media was withdrawn, replaced with fresh medium and analyzed spectrophotometrically at λmax= 303 nm using release media as blank43. The cumulative RES% released at different time intervals was determined as a percentage of the initial drug content. The release kinetics model was investigated using DDsolver software44. The study was conducted in triplicates, and the data were presented as mean ± SD.
Stability study
Blank LNCs, LIN-LNCs and LIN-LNC-RES formulations were stored at 4 °C for 3 months, and samples were collected at 0, 7, 30 and 60-day interval. Size, PDI, zeta potential and entrapment efficiency were measured to ensure stability over the test period compared to freshly prepared samples45.
Assessment of microbiological activity of the formulations
Microorganisms
The strains used in this study were Staphylococcus aureus (reference strain ATCC-6538) and 3 clinical isolates of Methicillin-resistant Staphylococcus aureus (MRSA) (obtained from the isolates repertoire at the Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University.
Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The strains were streaked for isolation onto nutrient agar plates, then one to two colonies were taken and suspended in 4 ml 0.9% NaCl. The turbidity of the microorganism suspension was adjusted to that of the 0.5 McFarland standard. The suspension was further diluted a 100-fold in double-strength Muller-Hinton Broth (MHB) medium to obtain a final bacterial inoculum of 106cfu/ml46.
The MIC value was determined by broth microdilution method as described previously46 with slight modifications. Briefly, serial 2-fold dilutions of the LNCs preparations in sterile water were prepared to obtain the desired concentration range. Aliquots of 100 µl of the dilutions or the control were dispensed in the wells of a 96-well microtiter plate. 100 µl of the bacterial suspension in double-strength MHB were added to each well. The positive control wells contained bacterial suspension in MHB broth without the tested sample. The negative control wells contained sterile MHB and the tested sample. The plates were then incubated for 24 h at 37 °C. The MIC was defined as the lowest concentration that completely inhibited the growth of the bacteria detected by the unaided eye.
MBC values were determined by transferring 5 µl from wells (of the MIC plate) showing no bacterial growth to a sterile 96-well plate containing 200 µl MHB and incubating for 24 h at 37 °C. MIC and MBC assays were performed in triplicate with different starting dilutions to determine the minimum inhibitory and bactericidal concentration for each tested preparation.
Time kill assay
The time kill assay against MRSA 3 was performed to assess the sustained effect of LIN-LNC-RES as an antibacterial nanosystem as previously described with slight modifications47. MRSA 3 was inoculated in MHB at a density of 108 cfu/ml alone (control) and in combination with LIN-LNC-RES at 0.25xMIC, 0.5xMIC, 1xMIC, 2xMIC and 4xMIC values and incubated in a shaker incubator at 120 rpm at 37 °C. Samples of the control group as well as treatment groups were withdrawn at 0, 3, 6, and 24 h and serially diluted in sterile saline. A 20 µl of each dilution was plated on nutrient agar and incubated for 18–24 h (Fig. 1a). The count was determined, multiplied by the used dilution factor and the rate of bacterial death was determined by plotting bacterial count (CFU/ml) against time.
Biofilm assays
Biofilm formation assay
Biofilm formation assay was conducted in a 96-well plate as previously described48 with slight modifications. Bacterial suspensions of the 3 MRSA isolates were cultured in brain heart infusion (BHI) supplemented with 1% glucose and normalized to an absorbance of 0.1 at 600 nm equivalent to 0.5 McFarland standard. 100 µl of the bacterial suspension (106 cfu/ml) were inoculated in each well either alone (control) or in combination with sub-MIC concentration range of the formulations. The plates were incubated for 24 h at 37 °C under static conditions, after which, the wells were decanted and washed with 1x PBS for three times then fixed with 2% formaldehyde. The adherent biofilm was stained with 200 µl 0.5% crystal violet (CV) for 30 min at room temperature. Excess stain was removed by washing three times with 200 µl 1x PBS and the plates were left to dry in an inverted position at room temperature overnight. The bound CV was solubilized using 95% ethanol in a shaking incubator for 1 h and quantified by measuring absorbance at OD595nm using an ELISA reader (Biotek, USA). Biofilm formation index (BFI) was used to evaluate biofilm formation ability in the presence and absence of the treatment using the following equation:
\(\:[\text{O}\text{D}\text{C}\text{V}\:\text{B}\text{i}\text{o}\text{f}\text{i}\text{l}\text{m}\:-\:\text{O}\text{D}\text{C}\text{V}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l})/\text{O}\text{D}\:\text{P}\text{l}\text{a}\text{n}\text{k}\text{t}\text{o}\text{n}\text{i}\text{c}]\)49.
Biofilm eradication assay
The biofilm eradication assay was conducted to study the ability of the formulations to disrupt previously formed biofilm50. 200 µl of the bacterial suspensions of the MRSA isolates were cultured in BHI supplemented with 1% glucose were pipetted in a 96-well plate and incubated for 48 h to allow biofilm formation. After incubation, the wells were decanted and different concentrations of the formulations were prepared in fresh broth and added in 100 µl aliquots to the plates then incubated for another 48 h. The rest of the protocol was done as described under the “biofilm formation assay” section.
Scanning Electron microscopy (SEM) study
SEM analysis of MRSA biofilm
The anti-biofilm activity of the formulations was assessed by SEM as previously described51 with slight modifications. The biofilm in absence (control) and presence of different treatments (RES, LIN, Blank LNC, LIN-LNC, LIN-LNC-RES) was grown on glass coverslips in a 6-well plate incubated at 37 °C for 24 h. The glass coverslips were then rinsed with saline and fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate-buffered saline. Samples were gradually dehydrated with ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100%) then dried and sputter coated with gold for SEM observation (JSM-IT200, JEOL, Japan).
SEM of bacterial membrane integrity
Alterations in the surface morphology and the cellular ultrastructure of the bacteria were investigated using SEM as previously described52 with some modifications. One representative clinical isolate of MRSA (MRSA-1) was added to Mueller Hinton broth (MHB) and adjusted to that of the 0.5 McFarland standard to obtain a bacterial inoculum of 106cfu/ml. The bacterial suspension was then incubated alone (control) or in combination with different treatments (RES, LIN, Blank LNC, LIN-LNC, LIN-LNC-RES) at a final concentration equivalent to the MIC for 24 h at 37 °C. After incubation, the samples were centrifuged at 6000 rpm for 10 min, decanted to remove excess liquid and a pellet was obtained and fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate-buffered saline (pH = 7.2). The cells were then gradually dehydrated using ethanol, and dried followed by sputter coating with gold for examination under SEM (JSM-IT200 Series, JEOL, Japan). The quantification of intact bacteria in SEM images was performed using ImageJ© software53.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Gene expression levels of a representative clinical MRSA isolate were analyzed using qRT-PCR as previously described54. The bacterium was grown alone or in the presence of RES or LIN-LNC-RES at their corresponding 0.5 MIC concentrations for 24 h, then centrifuged at 6000 rpm for 10 min and the supernatant layer was discarded. The pellets were then washed with 1x PBS once and stored at −80 °C. Total RNA was extracted using spin columns according to manufacturer’s instructions (Applied Biotechnology, Egypt) and its concentration and quality were assessed using the Nanodrop microvolume spectrophotometer (ThermoFisher, USA). Complementary DNA (cDNA) was synthesized from extracted RNA by reverse transcription using hexamer primers according to the manufacturer’s instructions (Applied Biotechnology, Egypt). qPCR was done using specific primers listed in Table 1 and SYBR Green master mix in a BioRad CFX96 real-time PCR system (BioRad, United States)55. Negative and positive controls were included in the analysis to normalize gene expression. Relative gene expression was analyzed using ΔΔCt values as compared to control. GyrA gene was used as an endogenous housekeeping control gene.
Cytotoxicity study
The effect of the LNCs on cell viability was determined by 3-(4, 5-Dimethyl-2-thizolyl)−2, 5 diphenyltertazolium bromide (MTT) assay on A549 cells56. A549 cells were maintained in DMEM high glucose (Biowest, France) supplemented with 10% Fetal Bovine Serum (FBS) (Biowest, France), 100U/ml penicillin and 1% streptomycin. Briefly, the cells were cultured in a 96-well plate at a density of 15,000 cells/well then treated with the treatment groups (RES, LIN, Blank, LIN-LNC and LIN-LNC-RES). After treatment for 24 h, the cells were incubated with MTT solution at a concentration of 0.5 mg/ml in PBS for 3 h at 37 °C. Following incubation, the supernatant layer was removed and DMSO was added to dissolve the formazan crystal. The absorbance was measured at OD490nm using an ELISA reader (Biotek, USA). Cell viability was calculated using the following equation:
Statistical analysis
All measurements were done in triplicates and presented as mean ± SD. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by a post-hoc Tukey test (GraphPad Prism version 9, CA, USA). The level of significance was determined at p ≤ 0.05.
Results
Preparation and characterization of LNCs
Blank LNC, LIN-LNC, and LIN-LNC-RES were prepared by PIT method. As shown in Table 2, the addition of LIN to the formulation (LIN-LNC) reduced the PIT significantly as compared to Blank LNC (82 °C vs. 25 °C). Incorporation of RES at an increasing concentration further reduced the PIT by 3 °C and 10 °C in case of formulations containing 1 & 2 mg/ml and 3 mg/ml respectively. The physicochemical properties of the prepared LNCs including size, PDI, zeta potential and % EE are illustrated in Table 2. Blank LNC had a size of 42.480 ± 0.729 nm which was significantly reduced after the addition of LIN in LIN-LNC resulting in a size of 37.923 ± 0.644 nm (p = 0.0013). LIN-LNC-RES at RES concentrations of 1, 2 and 3 mg/ml showed variable size, PDI and zeta potential as shown in Table 2. Among the formulations, LIN-LNC-RES (2 mg/ml) exhibited the most favorable physicochemical properties with a size of 35.193 ± 0.722 nm which was significantly lower than Blank LNC (p = 0.0003) and LIN-LNC (p = 0.0081). Similarly, LIN-LNC and LIN-LNC-RES (2 mg/ml) showed a narrow size distribution (Fig. 1a) with a significant reduction in PDI as compared to Blank LNC (p = 0.0205, p = 0.0069, respectively). Blank LNC had a negative zeta potential of −4.12 ± 0.187 which slightly decreased significantly in LIN-LNC and LIN-LNC-RES (−2.49 ± 0.127, −2.53 ± 0.065, respectively) (p = 0.0002). Due to the favorable physicochemical characteristics of LIN-LNC-RES (2 mg/ml), it was selected for further experiments. Entrapment efficiency % of RES in LIN-LNC-RES was determined by ultrafiltration/ultracentrifugation method and was found to be 98.09% ± 0.061 (Table 2) while drug content in 1 ml was calculated to be 1.97 ± 0.01 mg. TEM revealed the morphological characteristics of the different LNC formulations. As illustrated in Fig. 1b, Blank LNCs, LIN-LNCs, and LIN-LNC-RES all were spherical, homogeneously distributed, without any aggregation. These findings, demonstrate the successful formation of the LNCs with a well-defined and uniform spherical structure, regardless of the incorporation of the active compounds into the formulation.
Physicochemical characterization of Blank LNCs, LIN-LNC and LIN-LNC-RES.a: Size distribution, b: TEM images. Scale bars, 100 nm of i: Blank LNC, ii: LIN-LNC, iii: LIN-LNC-RES. c: DSC thermograms. Powdered samples were scanned at a heating rate of 10 °C/minute at and a temperature ranging from 25 to 300 °C. d: FT-IR analysis at a wavelength range between 4000 and 500 cm-1 at a resolution of 4 cm−1with an average of 100 scans. e: Release profile of RES from LNCs by dialysis method in PBS (pH 7.4) in a thermostatically controlled shaking water bath at 37 °C at 100 rpm (n = 3). f: Stability study of Blank LNC, LIN-LNC and LIN-LNC-RES at 4 °C over 3 months (n = 3).
DSC thermograms of free RES, LIN, physical mixtures 1 & 2, Blank LNCs, LIN-LNC, and LIN-LNC-RES were recorded to compare their thermal behavior. As shown in Fig. 1c, free RES showed a distinctive peak at 267.91°C confirming its crystalline structure. On the other hand, LIN exhibited 2 peaks at 116.47°C and 163.95°C, corresponding to oxidation and transition from liquid to vapor phases, respectively. After inclusion into the LNCs, RES and LIN peaks completely disappeared.
The intermolecular interactions between RES, LIN and LNCs components were studied by FTIR (Fig. 1d). Pure RES showed a band at 3200 ~ 3500 cm−1 which corresponds to the stretching vibrations of the phenolic –OH functional group. A narrow, low intensity band at 3009.37 ~ 2920.09 cm−1 reflects the unsaturated = C-H groups. The characteristic peaks at 1651.17 and 1435.54 cm−1 are associated with the stretching vibrations of the aromatic -C = C-. The peak at 1106.10 cm−1 corresponds with the olefinic C-C- stretching while the peak at 951.61 cm−1 is associated with the trans-olefinic bending alkene -C = C-. The vibrations of the arene functional group conjugated to the olefinic group if reflected by the peak at 796.93 cm−1. The incorporation of RES into the LNCs resulted in the reduction of the absorption intensities of its characteristic peaks indicating the formation of H-bonds and encapsulation into the hydrophobic core of the LNCs. In terms of LIN, an absorption peak was seen at 3431 cm−1 corresponding to the -OH stretching vibrations. The two absorption peaks at 3009.58 cm−1 and 2921.23 cm−1 correspond to the C-H aliphatic groups. A characteristic peak at 1650.52 correspond to the C = C stretching of the allyl group while the peaks at 1437.74 and 1019 cm−1 are associated with the C-O stretching bonds. The 3431 cm−1 peak was broadened after incoporation into the LNCs while the 3009.58 cm−1 and 2921.23 cm−1 were slightly shifted to a lower wavelength due to formation of hydrophobic bonds between LIN and LNCs components. Furthermore, the absorption peak of LIN at 1650.52 cm−1 shifted to a higher wavelength at 1743 cm−1.
The release profile of RES from LNCs investigated using dialysis method in PBS (pH = 7.4) is shown in Fig. 1e. The cumulative % released of RES encapsulated in LIN-LNC-RES was compared to the free drug. In case of free RES, 88.95% ± 0.585 was released after 2 hrs and > 99% was released after 24 h. On the other hand, RES release from LNCs showed a slow release profile over the 24 h with a total % release of 36.62% ± 0.035. The retarded cumulative release of RES from LNCs was statistically significant as compared to the free drug (p < 0.0001). The release of RES from LIN-LNC-RES followed Krosmeyer-Peppas kinetic model (R2 = 0.9135, AIC = 13.905, MSC = 2.0581).
All tested formulations (Blank LNCs, LIN-LNCs and LIN-LNC-RES ) showed good storage stability at 4°C with no significant changes in either size, PDI or zeta potential (Fig. 1f) or % EE over the study period (98.09 ± 0.061 vs. 98.06 ± 0.026 vs. 98 ± 0.021 vs. 98.03 ± 0.015, at 1 week, 1 month and 3 months respectively).
Assessment of microbiological activity of the formulations
The antibacterial activity of free RES, LIN, Blank LNCs, LIN-LNCs and LIN-LNC-RES was evaluated through the determination of MIC and MBC values against Staph. aureus (ATCC-6538) and 3 multi-drug resistant MRSA clinical isolates. As shown in Table 3, free RES had lower MIC values as compared to free LIN which was 27-22-fold lower than LIN in ATCC-6538 and the different MRSA isolates. In terms of free RES, MIC values (mg/ml) were the lowest in the standrard Staph. aureus ATCC-6538 (0.0625), followed by the more resistant different MRSA isolates (0.125–0.25 mg/ml).
Blank LNCs had no antibacterial activity against any of the strains. The incorporation of LIN into LNCs (LIN-LNCs) improved its antibacterial activity as demonstrated by reduced MIC values. LIN-LNCs MIC values were reduced 6.1-folds against the standard ATCC strain, and were reduced up to 3-folds against MRSA isolates.
In case of RES, encapsulation into LIN-LNC-RES also enhanced its antibacterial activity. LIN-LNC-RES had 4-fold decrease in MIC values against ATCC-6538, MRSA 1 and MRSA 3 while in case of MRSA 4, MIC was decreased by 2-folds only. These data suggests that MRSA 4 strain is more resistant than the other studied strains.
To further investigate the mechanism of antibacterial activity of the different treatments, whether they are bactericidal or bacteriostatic, the MBC values were determined (Table 3). Free RES exhibited bactericidal action against all isolates while free LIN was only bactericidal against ATCC-6538 and bacteriostatic against all MRSA isolates. LIN-LNCs exerted bactericidal action against ATCC-6538, MRSA 1 & 3, while they were shown to be bacteriostatic against MRSA 4 isolate. Interesitngly, LIN-LNC-RES showed extended bactericidal effect against MRSA 3 in addition to the standard staph. aureus strain, while it exerted bacteriostatic action against MRSA 1 and 4.
Time-kill assay
To ensure the sustained antibacterial activity of LIN-LNC-RES, the time-kill assay was conducted in MRSA 3 (Fig. 2a). As shown in Fig. 2b, LIN-LNC-RES exerted strong antibacterial action over 24 h period. There was no significant difference between control group and treatment group at 0.25x and 0.5x MIC at 3 h while at the MIC, 2x and 4x the MIC, there was significant reduction in bacterial counts (Fig. 2c). After 6 h, there was significant reduction in viable bacteria at all concentrations as compared to control group (p < 0.001). Similarly, at 24 h, significant antibacterial action was obsevered at all concentrations as compared to the control groups (p < 0.0001). Furthermore, there were significant differences between different concentrations of LIN-LNC-RES as shown in Fig. 2c.
Time-kill assay of LIN-LNC-RES over 24 h confirms the extended antibacterial activity of the nanosystem. a: A schematic diagram of time-kill assay procedures where MRSA 3 was allowed to grow in MHB then it was treated with LIN-LNC-RES at 0.25, 0.5, 1, 2 and 4x MIC value. Sample was withdrawn at time intervals (0, 3, 6 and 24 h), serially diluted then plated on agar to be incubated for 24 h at 37 °C. b: Time-kill assay of LIN-LNC-RES at different concentrations against control. c: Statistical analysis of LIN-LNC-RES at 0.25, 0.5, 1, 2 and 4x MIC demonstrates extended antibacterial activity as compared to control group. One-way ANOVA followed Tukey’s post-hoc test for multiple comparisons among different groups. * p < 0.05, ** P < 0.01, ** P < 0.001, **** p < 0.0001.
Biofilm assays
Biofilm combating assays were conducted to study the ability of the formulation to (1) prevent formation of biofilm (biofilm formation inhibition asssay) and (2) disrupt the already formed biofilm (biofilm detachment assay) in the tested MRSA clincal isolates. The IC50 values (for biofilm prevention and biofilm eradication) of the free compounds and the LNCs have been determined and are illustrated in Table 4 and supplementary Figs. 1 & 2.
Biofilm formation asssay
LIN-LNC-RES significantly improved the antibiofilm action of RES against all MRSA isolates as evident by significantly reduced IC50 values as shown in Table 4. Similarly, the antibiofilm action of LIN-LNC was significantly higher than free LIN against MRSA 3. On the contrary, LIN-LNC showed reduced biofilm prevention ability as compared to free LIN against MRSA 1 & MRSA 4 isolates. LIN-LNCs exerted significantly higher antibiofilm action when compared to Blank LNCs against MRSA 3. As compared to free LIN, free RES had significantly lower IC50 value against MRSA 4 (Table 4). Furthermore, LIN-LNC-RES showed an improved biofilm prevention activity as compared to LIN-LNCs against all MRSA isolates (Fig. 3a) (Supplementary Fig. 1a-c).
a: LIN-LNC-RES significantly inhibit biofilm formation among different MRSA clinical isolates compared to other treatment groups. Different MRSA isolates were allowed to form biofilm in the absence or in the presence of two fold serially diluted concentrations of the different treatment group (RES, LIN, Blank LNCS, LIN-LNC, LIN-LNC-RES). The biofilm mass was quantified using crystal violet and percentage inhibition of biofilm formation was calculated for each concentration relative to the untreated control. b: LIN-LNC-Res significantly eradicate formed biofilm among different MRSA clinical isolates compared to other treatment groups. First, different MRSA isolates were allowed to form biofilm then, the formed biofilm was left untreated or incubated with two fold serially diluted concentrations of the different treatment group (RES, LIN, Blank LNCS, LIN-LNC, LIN-LNC-RES). The biofilm mass was quantified using crystal violet and percentage biofilm eradication was calculated for each concentration relative to untreated control of each strain. Results shown a representative experiment of three independent experiments. One-way ANOVA followed Tukey’s post-hoc test for multiple comparisons among different groups. * p < 0.05, ** P < 0.01, ** P < 0.001, **** p < 0.0001.
Biofilm eradication assay
In biofilm detachment assay, LIN-LNC-RES demonstrated a significantly enhanced biofilm eradication activity as compared to free RES against all tested MRSA isolates (Fig. 3b). LIN-LNC significantly improved the antibiofilm action of free LIN against MRSA 3 and MRSA 4 as demonstrated by reduced IC50 values (Table 4). However, free LIN showed higher biofilm eradication as compared to LIN-LNC in case of MRSA 1. As compared to LIN-LNC, LIN-LNC-RES showed a significant improvement in biofilm eradication against all MRSA isolates (Fig. 3b). LIN-LNC had lower IC50 values as compared to Blank LNC against MRSA 1 and MRSA 3 (Table 4) (Supplementary Fig. 2a-2c).
Visualization of antibiofilm activity using SEM
The biofilm prevention activity of the free drugs and different formulas was then visualized using SEM at two magnification powers x5, 000 and x10, 000 (Fig. 4). As compared to control (Fig. 4a), free RES (Fig. 4b) and LIN (Fig. 4c) inhibited biofilm formation however, effect of RES was more pronounced than LIN. Blank LNC exerted little anti-biofilm activity as shown in Fig. 4d. The biofilm prevention activity of LIN-LNC was less than free LIN (Fig. 4e). On the other hand, LIN-LNC-RES exerted the most pronounced biofilm prevention effect among all evident by limited number of bacterial colonies shown in Fig. 4f. These results are in accordance with the IC50 value results.
LIN-LNC-Res significantly inhibit biofilm formation. SEM images of MRSA 1 biofilm after 24 h. incubation with different treatments at x5,000 and x10,000 magnification power. MRSA 1 were allowed to form biofilm on cover slips in the absence or in the presence of 0.5 MIC concentrations of the different treatment groups a: Control, b: RES, c: LIN, d: Blank LNC, e: LIN-LNC, f: LIN-LNC-RES. The biofilm mass was fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate-buffered saline then, gold sputtered and visualized.
Morphological changes in bacterial cell membrane
To visualize the effect of different formulations on the integrity of the bacterial membrane, SEM images were captured after incubation with the bacterial suspension. As shown in Fig. 5, as compared to the control groups (Fig. 5a), free RES disrupted the integrity of the bacterial membrane (Fig. 5b). Free LIN (Fig. 5c) had a negative impact on bacterial membrane but less pronounced than that of RES. On the other hand, Blank LNC had no effect on bacterial membrane integrity (Fig. 5d). LIN-LNC enhanced the effect of LIN as shown in Fig. 5e where pronounced destruction of bacteria was evident. LIN-LNC-RES exerted complete membrane destruction as shown in Fig. 5f confirming its powerful antibacterial activity as compared to other formulas. To confirm these results, differences in the antibacterial activity between treatment groups were statistically analyzed through the quantification of SEM images using ImageJ© software. As shown in Fig. 5g, the antibacterial activity of LIN-LNC-RES was the most pronounced amongst all treatment groups. Free RES had a significantly higher antibacterial action as compared to LIN. LIN-LNC improved the antibacterial activity of free LIN significantly. These results correlate well with MIC values as previously discussed.
LIN-LNC-Res significantly disrupt bacterial cell membrane integrity as compared to other treatment groups. SEM images of MRSA 1 bacterial membrane after 24 h. incubation with different treatments. a: Control, b: RES, c: LIN, d: Blank LNC, e: LIN-LNC, f: LIN-LNC-RES at x25,000. The bacterial cells were incubated with or without at MIC concentration of the treatment groups for 24 h. The samples were then centrifuged at 6000 rpm for 10 min, decanted and the pellet obtained was fixed with 4% formaldehyde and 1% glutaraldehyde in phosphate-buffered saline (pH = 7.2). The cells were then dehydrated using ethanol, dried followed by sputter coating with gold for examination under SEM. g: Quantification of viable bacteria in SEM images after treatment with different groups demonstrates superiority of LIN-LNC-RES.
Quantitative real-time polymerase chain reaction (qRT-PCR)
qRT-PCR was used to evaluate the functionality of the accessory gene regulator (agr) system and the two-component system SaeR/S. Bacterial cultures were allowed to grow in the absence or the presence of RES, or LIN-LNC-RES at their ½ MIC values for 24 h. Following incubation, RNA was extracted, and the expression levels of RNA III, SaeR and SaeS were determined (after normalization to the gyrA housekeeping gene). RES showed a significant decrease in RNA III expression level as compared to the untreated control (p = 0.0323). LIN-LNC-RES showed a significant reduction in RNA III expression level as compared to control (p = 0.0029) and free RES (p = 0.0146) (Fig. 6a). Similarly, RES significantly reduced the expression level of SaeR but not SaeS (Fig. 6b-c). LIN-LNC-RES reduced the expression levels of both SaeR and SaeS as compared to control group (p = 0.0073, p = 0.0060, respectively). On the other hand, LIN-LNC-RES exerted a more significant reduction in expression level of SaeS as compared to free RES but there was no significant difference in gene expression level of SaeR (Fig. 6b-c).
a-c: RES and LIN-LNC-RES downregulate the expression of different virulence factors. Shown are qPCR quantification results after treatment of MRSA 1 clinical isolate with RES, or LIN-LNC-RES. The bacterial cells were incubated with or without 0.5 MIC concentration of the treatment groups for 24 h. RNA was then extracted, and RT-qPCR of RNAIII,SaeR and SaeS was performed. Expression was normalized to gyrA housekeeping gene and represented a fold change compared to untreated control. a: RNAIII (a marker for agr activity), b: SaeR, c: SaeS. One-way ANOVA followed Tukey’s post-hoc test for multiple comparisons among different groups. * p < 0.05, ** P < 0.01. d: Schematic representation of the preparation of LNCs and their biological activity on S. aureus and MRSA clinical isolates.
Cytotoxicity study
The safety of Blank LNCs, LIN-LNC and LIN-LNC-RES was investigated in A549 lung cells using MTT assay. The incorporation of LIN in LNCs had a cytoprotective effect as shown in the significantly higher IC50 value as compared to Blank LNCs (p = 0.0115) (supplementary Fig. 3b, 3d). However, adding the cytotoxic RES to LIN-LNCs restored the cytotoxic potential of the system as shown in the lower IC50 values as compared to LIN-LNC (0.07896 mg/ml).
Discussion
Multi-drug-resistant MRSA is considered a life-threatening superbug responsible for a plethora of nosocomial and community infections57. According to the WHO, multi-drug-resistant MRSA has been stratified as high tier priority II pathogen leading to increased morbidity and mortality58,59. Clinically, several antibiotics are used to treat multi-drug-resistant MRSA infections such as vancomycin, linezolid, oritavancin, and dalbavancin however, resistance rapidly develops against these agents. Hence, the development of new antibacterial agents to subdue various resistance mechanisms is a crucial step to overcome MRSA infections. Essential oils and nanoparticles have been extensively studied as therapeutic alternatives to conventional antimicrobials and demonstrated promising ability to tackle antimicrobial resistance60.
In the current study, we introduced a novel linalool-based LNC system where linalool is incorporated as an integral component rather than an active constituent. Furthermore, linalool-based LNCs encapsulating the phytochemical RES were prepared by applying green nanotechnology principles through the PIT preparation method. The study also investigates, for the first time, the antibacterial and anti-virulence activity of the nanosystem against staphylococcus aureus including MRSA isolates (Fig. 6d).
In this study, the inclusion of LIN into the LNC formulation was intended to serve a dual purpose: first, as a bioactive molecule with well-reported antimicrobial effects, and second, to reduce the PIT below 50 °C, as the stability of RES is highly affected by temperature.
LIN resulted in a significantly reduced PIT as compared to Blank LNC. LIN is a monoterpenoid, a class of terpenes having the ability to reduce the phase transition temperature of lipids in a concentration-dependent manner61. Similarly, the encapsulation of RES further reduced the PIT as compared to both formulations. RES has been shown to influence thermotropic phase behavior of lipids in a concentration-dependent manner as well as demonstrated by Ceja-Vega et al.62. At low concentrations, RES positions itself near the head group region of lipids however, at higher concentrations, RES penetrates into the acyl region of lipids interacting with the hydrophobic chain. This resulted in a reduction in PIT of the lipid nanocapsules due to the change in the HLB value of lipids which directly influences the phase inversion temperature63,64.
Furthermore, LNC size decreased after incorporation of linalool mostly due to decreased oil to surfactant proportion, due to surface active property exerted by Linalool65,66. Encapsulation of RES into the LNCs further reduced its size which could be explained by the hydrophobic interactions between the drug and the LNC which was further verified in FT-IR analysis (Fig. 1d)62,63,64. Our results are similar to the findings of Jung et al.67. And Wan et al.68 where inclusion of RES into Poly (lactic-co-glycolic acid) (PLGA) NPs resulted in reduced particle size.
As for the zeta potential, Blank LNCs had higher slight negativity as compared to linalool-based LNCs which might be attributed to the formation of hydrophobic bonds between linalool and RES and the hydroxyl groups of the surfactant which were confirmed by FT-IR analysis (Fig. 1d).
TEM images of LIN-LNC-RES showed the same morphological characteristics for both Blank LNC and LIN-LNC, with no signs of separation of drug crystals or oil globules suggesting successful preparation and encapsulation of RES in the oily core of the LNCs. Similar results have been previously reported with quercetin69, Tanshinone IIA70and indomethacin/diclofenac71.
Effective encapsulation of RES (2 mg/ml) was demonstrated by high EE% which can be attributed to the high lipophilicity of RES in addition to the inherent property of LNCs to entrap lipophilic drugs in their oily core70,72.
DSC thermograms further confirmed RES encapsulation and its transition from the crystalline to the amorphous state evident by the disappearance of its characteristic peak as seen in RES nanosuspension investigated by Hao et al.73.
The release profile of LIN-LNC-RES showed a controlled and sustained release over 24 h. period as compared to free RES. The slow release of lipophilic drugs has been extensively studied in the literature. Owing to their high affinity, lipophilic drugs are usually embedded into the oily core of the LNCs in a soluble form, with a very slow release rate into the aqueous release medium.
Storage stability of Blank LNC, LIN-LNC and LIN-LNC-RES in 4 °C showed no change in the colloidal properties or EE% of any of the formulations proving the long-term stability of LNCs which has been addressed in the literature29,74,75. The stability of LNCs is due to the presence of the ionic surfactant ®in addition to the PEG moiety on the surface imposing steric hindrance thus preventing particle aggregation76.
The antibacterial activity of RES against gram-positive and gram-negative bacteria has been well documented in the literature21. Several mechanisms have been proposed for its antibacterial action such as ATP synthase inhibition, DNA fragmentation, up-regulation of SOS stress response regulon, suppression of ftsZexpression in addition to membrane damage through potassium leakage, and enhanced uptake of propidium iodide77,78,79. Several studies demonstrated the antibacterial activity of RES against S. aureuswith an MIC range of 100 - > 1000 µg/ml80,81,82. Our study showed that RES had a prominent antibacterial activity against S. aureusand MRSA isolates. However, reduced antibacterial activity and development of resistance may occur due to active extrusion of RES by efflux pump83,84. Encapsulation of RES into the LNCs remarkably improved their antibacterial activity as evident by reduced MIC values. This can be explained by the improved permeation of LNCs through bacterial membranes which enhance the delivery of RES15,85,86. Moreover, owing to their Kolliphor content; LNCs’ inherent ability to inhibit efflux pumps leads to increased accumulation and prolonged contact time between the drug and the bacteria and reduce the development of resistance85,87. Furthermore, the LNCs themselves have inherent antibacterial activity due to the presence of capric/caprylic triglycerides in their composition88. Similarly, the inclusion of RES into biogenic silica NPs enhanced its antibacterial and antibiofilm activity against S. aureus89. However, they are contrary to Prevete et al.90 where encapsulation of RES into liposomes didn’t induce any antibacterial activity as compared to free RES. MBC results showed that free RES was bactericidal against S. aureus and MRSA isolates. Yet, after encapsulation into LNCs, LIN-LNC-RES extended its bactericidal activity against S. aureus and MRSA 3 while it was bacteriostatic against MRSA 1 and 4. This could be explained by inter-strain variability leading to different antibacterial responses. The sustained antibacterial activity of LIN-LNC-RES was demonstrated by the time-kill assay where the slow controlled release of RES from the LNCs didn’t interfere with its antibacterial action over 24 h. Our results are in agreement with the study by Spósito et al.91 where encapsulation of RES into chitosan nanoparticles improved its antibacterial activity over 24 h.
The antibacterial activity of linalool has been investigated in several studies against Pseudomonas aeruginosa92, Listeria monocytogenes93, Shigella sonnei94, and Campylobacter spp.33among others. The proposed mechanism is through the inhibition of bacterial cell wall33,95. However, its practical use is hindered by its volatility and low water solubility which we overcame using LIN-loaded nanocarriers. Our study showed that linalool had antibacterial activity against S. aureus and MRSA isolates. However, the antibacterial action was less prominent compared to RES. Incorporating LIN into LNCs improved its antibacterial activity. These results are in line with Jin et al.96 where encapsulation of LIN into mesoporous silica improved its antibacterial activity. In addition, Taghavi et al.97 proved that LIN nanoemulsions have enhanced antibacterial activity as compared to free LIN. MBC values showed there was no difference in the mechanism of antibacterial action between free LIN and LIN-LNCs with both being bacteriostatic against MRSA isolates and bactericidal against S. aureus.
The antivirulence properties of RES have been well studied in the literature and include antibiofilm properties, antimotility properties, interference with toxin production and quorum sensing, reduced adhesion and colonization21. Biofilm formation affects the successful management of infectious diseases and is a main cause of chronic and recurrent infections. Biofilms are also an important cause of antimicrobial resistance as they protect the planktonic bacteria from external environmental challenges, reduce phagocytosis and prevent the penetration of antimicrobial agents98. The antibiofilm properties of RES have been investigated on several bacterial pathogens including gram-positive and gram-negative bacteria99,100,101. Several mechanisms are involved in the antibiofilm activity of RES which include the down-regulation of certain genes which play a regulatory role in biofilm formation such as the ABC transporter substrate-binding protein (FN0659)102. Another mechanism is through the inhibition of quorum sensing (QS) which plays a critical role in biofilm formation102. Furthermore, RES was shown to inhibit ZraS/ZraR signaling and to disrupt Zn ions balance inside and outside bacterial cells which in turn deregulate Glgs thereby inhibiting biofilm formation103. Lastly, RES was found to inhibit pyruvate dehydrogenase resulting in reduced levels of pyruvate which leads to inhibition of biofilm formation and its disruption103. Our study investigated the potential of RES to inhibit and/or eradicate the biofilms of different MRSA strains. Free RES demonstrated a biofilm prevention activity against all MRSA strains with varying IC50s due to variations between strains. However, in the biofilm eradication assay, RES showed antibiofilm activity against MRSA 1 and MRSA 3 strains but not MRSA 4 which can be associated with strain variations where MRSA 4 showed the most resistant phenotype. After encapsulation of RES into LIN-LNCs, the antibiofilm activity significantly improved as demonstrated by reduced IC50values against all strains. This can be attributed to the ability of LIN-LNCs to easily penetrate through the biofilm and deliver the drug effectively in addition to its ability to inhibit efflux pumps85,87. Similar results were obtained by Aiello et al., where encapsulation of RES in NPs improved its antibiofilm activity104.
The antibiofilm properties of LIN against several pathogenic bacteria are well documented in the literature33,105,106. The antibiofilm activity of LIN is mainly due to the interaction between hydroxyl (OH−) groups in linalool with positively charged bacterial cell surface molecules, altering bacterial hydrophobicity and cell charge which is crucial for its adhesion and biofilm initiation107. Moreover, in gram-positive bacteria, linalool was found to down-regulate dltAgene which is responsible for cell adhesion and biofilm initiation106. Furthermore, LIN was found to bind to histidine kinase ComP and TasA proteins suppressing bacterial motility and the production of extracellular polysaccharides which are the main components of biofilms106. Our study showed that LIN could prevent biofilm formation at IC50s comparable to free RES. In the biofilm eradication assay, LIN showed antibiofilm activity against MRSA 1 and MRSA 3 but not MRSA 4 which was also comparable to results with RES. However, the antibiofilm activity of RES was significantly more pronounced than LIN against MRSA 4 in case of biofilm prevention, and against MRSA 1 in case of biofilm eradication. Incorporation of LIN into LNC structure showed a variability in antibiofilm activity against MRSA isolates. LIN-LNCs had significantly lower biofilm prevention activity against MRSA 1 and MRSA 4 while an improvement in biofilm prevention was observed in case of MRSA 3. On the contrary, in biofilm eradication assay, LIN-LNC had lower antibiofilm activity against only MRSA 1 while a significant improvement was seen in case of MRSA 3 and MRSA 4. A possible explanation of the reduced antibiofilm activity is due to the unavailability of OH− groups on linalool after incorporation into the LNCs which was confirmed by FT-IR. Thus, LIN can no longer interact with positively charged cell surface molecules on bacterial cells to prevent their adhesion and biofilm formation.
To better visualize the interaction between free drugs, the formulas and the bacterial membrane, SEM images were taken after incubation with the bacteria for 24 h. As shown in Fig. 5, free RES and LIN disrupted the membrane of the bacteria as compared to the control group. LIN-LNC effect was more pronounced than free LIN and similarly, LIN-LNC-RES membrane disruption was significantly higher than free RES. Our results are similar to those of Song et al.108 and Han et al.109 who investigated the antibacterial activity of EOs on S. aureus. Moreover, Dhanam et al.110 investigated the antibacterial activity of free bacteriocin versus bacteriocin encapsulated in silver nanoparticles against MRSA and found that encapsulation into NPs improved the antibacterial activity as compared to the free drug.
We then visualized the antibiofilm activity of free RES, LIN, Blank LNC, LIN-LNC and LIN-LNC-RES using SEM at two magnification powers x5000 and x10,000. As shown in Fig. 3, free RES and LIN could prevent biofilm formation as compared to control. Our results are similar to Aiello et al.104 who also investigated the antibiofilm activity of RES against MRSA. Blank LNC and LIN-LNC had some biofilm prevention activity but not as pronounced as the free drugs. However, LIN-LNC-RES showed the most biofilm prevention activity among all treatment groups which is similar to Aiello et al.104 results where the inclusion of RES into nanoparticles enhanced its antibiofilm activity.
Biofilm formation process by the clinical MRSA isolates was reported to be ica-independent involving the agr and staphylococcal accessory regulator A (sarA) regulatory mechanisms111. The agr system is a part of the quorum sensing (QS) system that regulates biofilm formation and is composed of agrA and regulatory RNAIII111. RNAIII is a post-transcriptional regulator of multiple virulence genes whose expression levels can be used as an indicator for the agr system functionality. These genes are up regulated in MRSA infections and are responsible for its virulence. Thus, antimicrobial agents which have the ability to down-regulate the agrsystem can inhibit biofilm formation and reduce MRSA virulence112. In this study, the ability of RES to reduce the expression of the RNAIII was investigated using qRT-PCR analysis. Results showed that RES significantly down-regulated RNAIII expression which is similar to Qin et al.113results which showed significant reduction in gene expression. Encapsulation of RES into LNCs (LIN-LNC-RES) showed a significant reduction in gene expression as compared to control and free RES. This may be explained by the fact that LIN-LNCs improved the delivery of RES into the bacteria in addition to inhibiting resistance mechanisms such as efflux pumps85,87. These qPCR data suggest that the inhibition of the agr quorum sensing system is another potential mechanism by which LIN-LNC-RES exert their antibacterial and anti-virulence effects. The SaeRS system is a two-component regulatory system of S. aureus affecting the expression of many genes. It acts synergistically with sarA to promote biofilm formation. They both enhance the transcription of fibronectin binding protein A & B (fnbA, fnbB) and other adhesins which play a critical role in biofilm formation114. Furthermore, an interrelated regulation occurs between RNAIII and SaeRSwhich also plays a role in biofilm formation115,116. Thus, we investigated the impact of free RES and LIN-LNC-RES on the expression levels of SaeR and SaeS. RES reduced expression levels of SaeR and SaeS however, significant reduction was seen only with SaeR. Our results are similar to Duan et al.117 where RES reduced the levels of SaeRS at different MIC concentrations, however, the extent of reduction was different between isolates. Encapsulation of RES into LNCs significantly improved its activity on gene expression as compared to free RES in case of SaeS but not in case of SaeR. The observed differences in RES and LIN-LNC-RES effect on gene expression of SaeR and SaeS require further investigation to understand the mechanism underlying these differences.
The safety and biocompatibility of LNCs were investigated by MTT assay in A549 lung cells and showed reduction in cell viability with IC50 values of 0.04673 (204.7317 µM), 0.07896 (345.9365 µM) and 0.008938 mg/ml (39.2 µM) for the Blank LNCs, LIN-LNC and LIN-LNC-RES, respectively. Thus, as compared to LIN-LNC-RES, LNCs as a delivery system is considered relatively safe and biocompatible. These results are in accordance with the study by Le Roux et al.118that showed that LNCs exhibit cytotoxicity at a concentration of 2.27 mg/mL, 1.74 mg/mL and 0.0827 mg/mL for 25, 55 and 100 nm LNCs. The incorporation of LIN into LNCs improved its safety and biocompatibility as compared to the Blank LNC. LIN is a safe, biocompatible essential oil which has been used as an antibacterial, anti-inflammatory, anti-cancer, among other biomedical applications33,94,119. The utilization of LIN as an integral component of lipid nanoparticles have been investigated in several studies and proved the biocompatibility of LIN in vitro and in vivo120,121. The increased cytotoxicity of LIN-LNC-RES is due to the fact that RES is a potent anticancer compound against several types of cancer122,123,124. The investigation of biocompatibility of LNCs on 2D cell culture doesn’t reflect their true safety aspects. To obtain more reliable results on the safety and biocompatibility of LNCs, 3D cell cultures or in vivo toxicological assessment of major organs are required. In the study by Gaber et al.125, Blank LNCs showed no toxicity on the liver and didn’t affect the survival of rats. Thus, LNCs either conventional (Blank) or LIN-LNC can be considered a relatively safe, biocompatible nanosystem however, further studies to evaluate the safety of LIN-LNC-RES as an antibacterial system are required to reach a definitive conclusion.
Study limitations and future perspectives
The current study investigated the antibacterial, antibiofilm and anti-virulence efficacy of LIN-LNC-RES formulated by applying the principles of green nanotechnology. It demonstrated the superiority of LIN-LNC-RES over LIN-LNC and free drugs against S. aureusand MRSA clinical isolates. However, there are several limitations that need to be addressed in order to enhance its applicability and clinical translation. Generally, there was consistency in the findings among the tested strains, however the small number of tested clinical isolates somewhat limits the generalizability of the results. Although LNCs are prepared by PIT methods which is considered an eco-friendly approach involving less production steps, the success of scalability of this nanosystem should be examined. Although several synthesis parameters such as ratio of solutol: labrafac: linalool, concentration of RES, stirring rate and temperature have been tested to optimize the final formulation, further investigation of the effect of modifying these parameters on the physicochemical properties in addition to the biological efficacy of the nanosystem is warranted to give in-depth understanding and better optimization. The stability on LNCs in serum and in the GIT has been confirmed in several studies126,127,128 yet the impact of these variables on the drug in terms of stability and efficacy is required. 2D-cytotoxicity studies were conducted on A549 lung cells, however, in vivo toxicological studies on major organs such as lungs, liver and kidney to assess the safety and biocompatibility of LNCs haven’t been performed.
Conclusion
AMR rates are increasing dramatically worldwide, where MRSA contributes to increased morbidity and mortality. To date, antibiotics and antibiotic combinations don’t provide optimal management of MRSA infections. Nowadays, alternative therapeutic approaches based on nutraceuticals and nanoparticles have gained a lot of interest. In the current study, linalool-based LNCs loaded with RES were prepared by an environmentally-friendly PIT method and their antibacterial and anti-virulence activity against MRSA were assessed. LIN-LNC-RES demonstrated favorable physicochemical characteristics in terms of colloidal properties, high entrapment efficiency, stability over 3 months and a controlled drug release profile. The antibacterial and antibiofilm activity of LIN-LNC-RES was confirmed by MIC and IC50 values in addition to SEM images. Moreover, LIN-LNC-RES exerted anti-virulence activity by interfering with the gene expression of RNAIII potentially affecting quorum sensing as well as affecting SaeRS two component system (major regulator of virulence factors). Thus, our study provides proof-of-concept that LIN-LNC-RES can be used as a potent antimicrobial against MRSA to overcome antimicrobial resistance.
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Funding
This study was partly supported by the German Research Council (DFG) through grant ZI665/3 − 1 to Alaa Abouelfetouh. The work also received financial support from ICGEB and COMSTECH (Committee on Scientific and Technological Cooperation of the Organization of Islamic Cooperation), project No. CRP/EGY23-01.
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“All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Gaidaa Dogheim, Michael Shehat and Dina Mahdy. The first draft of the manuscript was written by Gaidaa Dogheim and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”
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Dogheim, G.M., Shehat, M.G., Mahdy, D.M. et al. Antibacterial and anti-virulence activity of eco-friendly resveratrol-loaded lipid nanocapsules against methicillin-resistant staphylococcus aureus. Sci Rep 15, 14677 (2025). https://doi.org/10.1038/s41598-025-95343-w
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DOI: https://doi.org/10.1038/s41598-025-95343-w
