biomedicines

Article Preparation of Terpenoid-Invasomes with Selective Activity against S. aureus and Characterization by Cryo Transmission Electron Microscopy

Bernhard P. Kaltschmidt 1, Inga Ennen 1, Johannes F. W. Greiner 2 , Robin Dietsch 3, 3 2,4 2, 1, , Anant Patel , Barbara Kaltschmidt , Christian Kaltschmidt † and Andreas Hütten * † 1 Thin Films & Physics of Nanostructures, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany; [email protected] (B.P.K.); [email protected] (I.E.) 2 Department of Cell Biology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany; [email protected] (J.F.W.G.); [email protected] (B.K.); [email protected] (C.K.) 3 Fermentation and Formulation of Biologicals and Chemicals, Bielefeld University of Applied Sciences, Interaktion 1, 33619 Bielefeld, Germany; [email protected] (R.D.); [email protected] (A.P.) 4 Molecular Neurobiology, Bielefeld University, Universitätsstrasse 25, 33615 Bielefeld, Germany * Correspondence: [email protected]; Tel.: +49-521-106-5418 These authors contributed equally to this work. †


Received: 7 April 2020; Accepted: 30 April 2020; Published: 1 May 2020


Abstract: Terpenoids are natural plant-derived products that are applied to treat a broad range of human diseases, such as airway infections and inflammation. However, pharmaceutical applications of terpenoids against bacterial infection remain challenging due to their poor water solubility. Here, we produce invasomes encapsulating thymol, menthol, camphor and 1,8-cineol, characterize them via cryo transmission electron microscopy and assess their bactericidal properties. While control- and cineol-invasomes are similarly distributed between unilamellar and bilamellar vesicles, a shift towards unilamellar invasomes is observable after encapsulation of thymol, menthol or camphor. Thymol- and camphor-invasomes show a size reduction, whereas menthol-invasomes are enlarged and cineol-invasomes remain unchanged compared to control. While thymol-invasomes lead to the strongest growth inhibition of S. aureus, camphor- or cineol-invasomes mediate cell death and S. aureus growth is not affected by menthol-invasomes. Flow cytometric analysis validate that invasomes comprising thymol are highly bactericidal to S. aureus. Notably, treatment with thymol-invasomes does not affect survival of Gram-negative E. coli. In summary, we successfully produce terpenoid-invasomes and demonstrate that particularly thymol-invasomes show a strong selective activity against Gram-positive bacteria. Our findings provide a promising approach to increase the bioavailability of terpenoid-based drugs and may be directly applicable for treating severe bacterial infections such as methicillin-resistant S. aureus.

Keywords: terpenoids; invasomes; thymol; menthol; camphor; cineol; S. aureus; E. coli; bactericidal

1. Introduction Terpenes are secondary plant metabolides with aromatic characters found in the oil fraction of various plants, where they serve for protection against predators or pathogens [1,2]. Notably, the oxygenated derivatives of terpenes, so-called terpenoids, have strong antioxidant and anti-inflammatory as well as antimicrobial properties [3,4]. For instance, the terpenoid thymol was reported to attenuate allergic airway inflammation in mice [5] and inhibit lipopolysaccharide-stimulated

Biomedicines 2020, 8, 105; doi:10.3390/biomedicines8050105 www.mdpi.com/journal/biomedicines Biomedicines 2020, 8, 105 2 of 18 inflammatory responses via down-regulation of the transcription factor NF-κB[6]. Additionally, the terpenoid cineol was shown to inhibit pro-inflammatory signaling mediated by NF-κB[7], while simultaneously potentiating IRF3-mediated antiviral responses [8]. In addition, cineol reduced production of mucus in a human ex vivo model of late rhinosinusitis [9]. Regarding the antimicrobial properties of terpenoids, thymol was reported to have direct bactericidal effects against S. aureus and S. epidermidis [10]. The terpenoids menthol and camphor also showed anti-bacterial activity against different bacterial species such as streptococci or mycobacteria [11,12]. Cineol was also recently demonstrated to display antibacterial activities against pathogenic bacteria present in chronic rhinosinusitis such as S. aureus [13]. Despite these promising anti-inflammatory and anti-bacterial properties, pharmaceutical applications of terpenoids against bacterial infection remain challenging due to their poor water solubility and high volatility. Here, we address this challenge by utilizing liposomal packaging for drug delivery of terpenoids. Liposomes are spherical vesicles of phospholipid bilayers, which are commonly used for encapsulation of drugs and particularly for increasing or allowing their anti-microbial activity [14]. For instance, Aravevalo and colleagues showed an increase in antibiotic activity of ß-Lactam against resistant S. aureus after encapsulation within coated-nanoliposomes [15]. In addition, Moyá and coworkers recently demonstrated a bactericidal activity of Cefepime encapsulated into cationic liposomes against E. coli [16]. Engel and colleagues assessed the antimicrobial activity of thymol and carvacrol encapsulated into liposomes by thin-film hydration and observed an inhibition of S. aureus and S. enterica growing on stainless steel [17]. As recently reported by Usach and coworkers, pompia essential oil (containing limonene and citral) as well as citral itself were successfully loaded into liposomes via hydration followed by ultrasonic disintegration. The respective encapsulated terpenoids had antimicrobial properties against different bacterial species such as S. aureus or P. aeruginosa [18]. A nanoemulsion comprising eucalyptus oil obtained by ultrasonic emulsification was also shown to have antibacterial activity against S. aureus [19]. Cui and colleagues further observed an antibacterial effect of cinnamon oil (with eugenol being its main compound) encapsulated into liposomes against methicillin-resistant S. aureus (MRSA) alone or cultivated as a biofilm [20]. In addition to increasing the anti-microbial activity of drugs, encapsulation into liposomal structures was already described to enhance the anti-inflammatory capacity of terpenoids. In this regard, nanostructured lipid carriers encapsulating thymol were shown to provide a sustained release of the terpenoid as well as an increase in anti-inflammatory activity within mouse models of skin inflammation [21]. In addition to nanostructured lipid carriers, terpenoids can also be delivered by encapsulation into polymeric nanostructured systems or by molecular complexation [2]. In addition to increasing stability of encapsulated compounds, terpenoid-encapsulation systems are widely accepted to be non-cytotoxic and enhance the antioxidant and anti-inflammatory activities of terpenoids ([18,21–23] reviewed in [2,24]). For instance, Manconi and colleagues reported the liposomal formulation of thymus essential oil to be highly biocompatible and to counteract oxidative stress in keratinocytes [22]. Thymol encapsulated in nanostructured lipid carriers further showed anti-inflammatory activity in different mouse models of skin inflammation in vivo [21]. In the present study, we took advantage of a liposome-based system, the so-called invasomes, for encapsulating the terpenoids thymol, menthol, camphor and cineol (Figure1). Invasomes are liposomes composed of unsaturated phospholipids, small amounts of ethanol, terpenes and water [25]. In our present approach, terpenoid-invasomes were produced via extrusion of a solution comprising the respective terpenoid solved in ethanol, while soybean lecithin served as a lipid source. We aimed to characterize the produced invasomes in terms of their lamellarity, size and bilayer thickness using cryo transmission electron microscopy (Cryo TEM). Although being commonly utilized for the formulation of invasomes [26,27], terpenoids mostly serve as permeation enhancer for transdermal delivery and bioavailability rather than being bioactive components [2]. In the present study, we focused on the production of invasomes with terpenoids as the bioactive components to inhibit bacterial cell growth. Biomedicines 2020, 8, 105 3 of 18

With respect to pharmaceutical applications for treating bacterial infection, we therefore assessed Biomedicinespotential 2020 bactericidal, 8, x FOR PEER effects REVIEW of our produced terpenoid-invasomes against S. aureus and E. coli3. of 18

FigureFigure 1. 1. StructuralStructural formulas formulas of of the the terpenoids terpenoids thymol, thymol, menthol, menthol, camphor camphor and and cineol cineol used used for for encapsulationencapsulation into into invasomes. invasomes.

2.2. Materials Materials and and Methods Methods

2.1.2.1. Preparation Preparation of of Invasomes Invasomes and and Encapsulation Encapsulation of of Terpeonoids Terpeonoids ForFor production production of of invasomes, invasomes, 20 20 mg mg of ofthe the respective respective terpenoid terpenoid thymol, thymol, menthol, menthol, camphor camphor or cineolor cineol (Caesar (Caesar and andLoretz Loretz GmbH, GmbH, Hilden, Hilden, Germany) Germany) was solved was solved in 1.32 in mL 1.32 ofmL 99.6% of 99.6%Ethanol. Ethanol. This mixtureThis mixture was subsequentlywas subsequently added added to 40 to mL 40 mLof 0.9% of 0.9% saline saline to reach to reach a final a final concentration concentration of of 0.5 0.5 mg mg terpenoidterpenoid per per mL. AA quantityquantity of of 100 100 mg mg of of soybean soybean lecithin lecithin (Lipoid (Lipoid S 100, S 100, Batch Batch 579000-1160713-01 579000-1160713-/704, 01/704,Lipoid Lipoid GmbH, GmbH, Ludwigshafen Ludwigshafen am Rhein, am Germany)Rhein, Germany) was added was to added 2 mL ofto this 2 mL solution of this comprising solution comprisingthe terpenoid, the terpenoid, ethanol and ethanol saline and followed saline byfollo vortexingwed by vortexing for 5 min for to 5 reach min to a homogenousreach a homogenous solution. solution.For control For formulation,control formulation, a solution a solution without withou terpenoidst terpenoids was applied.was applied. Invasomes Invasomes were were formed formed by byextrusion extrusion using using the the Avanti Avanti Mini-Extruder Mini-Extruder (Avanti (Avant Polari Polar Lipids, Lipids, Alabaster, Alabaster, AL, AL, USA) USA) by by passing passing 1 mL 1 mLof finalof final solution solution 10 times10 times back back and and forth forth through through a polycarbonate a polycarbonate membrane membrane with with 100 100 nm nm pores. pores.

2.2.2.2. Cryo Cryo Transmission Transmission Electron Electron Microscopy Microscopy (Cryo (Cryo TEM) TEM) µ ForFor Cryo Cryo TEM TEM analysis, analysis, 3 3µLL of of the the respective respective invasome-dispersion invasome-dispersion produced produced as as described described above above waswas placed onon aa TEM TEM copper copper grid grid (Quantifoil (Quantifo Microil Micro Tools GmBH,Tools GmBH, Großlöbichau, Großlöbichau, Germany). Germany). Plunging Plunginginto liquid into ethane liquid using ethane Leica using EM GPLeica (Leica EM Microsystems,GP (Leica Microsystems, Wetzlar, Germany) Wetzlar, with Germany) 80% moisture, with 80% 10 s moisture,pre-blotting 10 s time, pre-blotting 3 s blotting time, time 3 s andblotting 20 ◦ Ctime temperature and 20 °C wastemperature followed was by transporting followed by thetransporting samples to thethe samples cryo transfer to the stationcryo transfer (Fischione station Intruments, (Fischione Export, Intruments, PA, USA) Export, in liquidPA, USA) nitrogen. in liquid Analysis nitrogen. was Analysisdone at thewas OWL done Analytic at the OWL Center Analytic using Jeol Center JEM 2200using FS Jeol (JEOL JEM Ltd., 2200 Tokyo, FS (JEOL Japan) Ltd., operated Tokyo, at Japan) 200 kV. operated at 200 kV. 2.3. Determination of Zeta Potentials

2.3. DeterminationZeta potentials of Zeta were Potentials measured using Beckmann Coulter Delsa Nano C Particle Analyzer (Beckman Coulter, Brea, CA, USA) in a flow cell after dilution of samples with water from 50 mg/mL to 500 µg/mL. Zeta potentials were measured using Beckmann Coulter Delsa Nano C Particle Analyzer Measurements were repeated ten times. (Beckman Coulter, Brea, CA, USA) in a flow cell after dilution of samples with water from 50 mg/mL to2.4. 500 Evaluation µg/mL. Measurements of Invasome Size were and repeated Bilayer Thickness ten times.

2.4. EvaluationArea and of bilayer Invasome thickness Size andof Bilayer produced Thickness invasomes were measured using FIJI [28] by utilizing Cryo TEM images. Briefly, the area of every invasome was marked with the circular selection tool and the measurementArea and bilayer function thickness was appliedof produced to calculate invasome thes areawere of measured the selections using followed FIJI [28] by by calculation utilizing Cryoof the TEM radius. images. For Briefly, assessing the bilayers area of thickness,every invasome the segmented was marked area wi selectionth the circular tool of selection FIJI was tool used andfollowed the measurement by the straighten function function was of applied FIJI to obtainto calculate straight the selection area of and the calculation selections of followed a line profile. by calculationRespective of line the profiles radius. showed For assessing a clear dentbilayers at the thickness, bilayer position the segmented and thickness area selection was measured tool of at FIJI half wasmaximum. used followed A Gauss by distribution the straighten was function fitted to of all FIJI histograms. to obtain straight selection and calculation of a line profile. Respective line profiles showed a clear dent at the bilayer position and thickness was measured at half maximum. A Gauss distribution was fitted to all histograms.

2.5. Growth-Inhibition Zone Assay Overnight suspension cultures of S. aureus (Staphylococcus aureus Rosenbach 1884, DSM 24167, German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany) were inoculated on Brain Heart Infusion Broth (BHI) agar plates (Sigma-Aldrich Corporation, Merck

Biomedicines 2020, 8, 105 4 of 18

2.5. Growth-Inhibition Zone Assay Overnight suspension cultures of S. aureus (Staphylococcus aureus Rosenbach 1884, DSM 24167, German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Braunschweig, Germany) were inoculated on Brain Heart Infusion Broth (BHI) agar plates (Sigma-Aldrich Corporation, Merck KGaA, Darmstadt, Germany). Filter plates loaded with 10 µL of respective invasome-dispersion were placed on the BHI agar plates followed by incubation overnight at 37 ◦C. Diameters of the growth-inhibition zones were measured and calculated using FIJI [28].

2.6. Analysis of Encapsulation Efficiency and Loading Capacity Encapsulation efficiency (EE %) was analyzed as described in [29]. EE% was calculated by (total drug added free non-entrapped drug) divided by the total drug added. Loading capacity (LC) was − calculated as the amount of drug loaded per unit weight of total invasomes (weight of lipids). Free drug was separated from invasome preparation by ultrafiltration (1000 g for 30 min at 4 C) using an × ◦ Amicon Centricon device with a molecular weight cut-off of 30,000. Drug stocks for measurement were prepared in methanol for HPLC (VWR) at a concentration of 1 mg/mL. A linear dilution was prepared in methanol and absorbance was measured at 276 nm using an Ultrasspec 2100 pro photometer (Amersham Biosciences, Little Chalfont, UK). Linearity was proven between 0.0015 mg/mL and 0.025 mg/mL thymol. EE% for thymol was calculated as (0.5 mg/mL 0.0041 mg/mL)/0.5 mg/mL = 0.9918 resulting − in 99.18% EE. LC% was calculated as (0.5 mg/mL 0.0041 mg/mL)/(50 + 0.5) mg/mL= 0.01, which − equals 1%.

2.7. Flow Cytometric Measurement of Cell Death For flow cytometric measurement of cell death, S. aureus or E. coli (Escherichia coli DH5-Alpha) were exposed to respective invasome-dispersions (0.5 mg/mL, 1 mg/mL, 2 mg/mL final terpenoid concentration) for 24 h. Cells were fixed with 0.4% PFA for 20 min followed by staining with 1 µL/mL propidium iodide (PI, Sigma Aldrich) for 10 min. PI-stained bacterial cultures were analyzed using a Beckman Coulter Gallios Flow Cytometer (Beckman Coulter) followed by data analysis with Kaluza software (Beckman Coulter).

2.8. Statistical Analysis For assessment of lamellarity and size of invasomes encapsulating a distinct terpenoid, up to 200 invasomes per Cryo TEM image were measured in 3–4 representative Cryo TEM images. For evaluation of bilayer thickness, up to 30 invasomes were measured in 3–4 representative Cryo TEM images for each of the different terpenoid-invasomes. Examples of small representative sections of original cryo electron micrographs used for measuring invasomes size are included within the respective figures, details of measuring are described above. Statistical analysis was performed using Graph Pad Prism (GraphPad Software, San Diego, CA, USA). The p value is a probability, with a value ranging from zero to one. The first step is to state the null hypothesis, here that the terpenoids do not affect the size of the invasomes and all differences in size are due to random sampling. The p-value is the probability of obtaining results as extreme as the observed results of a statistical hypothesis test, assuming that the null hypothesis is correct. The p-value is used as an alternative to rejection points to provide the smallest level of significance at which the null hypothesis would be rejected. A smaller p-value means that there is stronger evidence in favour of the alternative hypothesis. For analysis of lamellarity, * p < 0.05 was considered significant (Mann–Whitney test, one-tailed). For analysis of invasome size, p < 0.0001 was considered significant (unpaired t-test, one-tailed). Growth-inhibition zone assay was performed as biological triplicate and Graph Pad Prism served for statistical analysis with * p < 0.05 being considered significant (Mann–Whitney test, one-tailed). Biomedicines 2020, 8, 105 5 of 18

3. Results and Discussion

3.1. Succsessfull Preparation of Invasomes Encapsulating the Terpenoids Thymol, Menthol, Camphor and Cineol

BiomedicinesTo encapsulate 2020, 8, x FOR terpenoids PEER REVIEW into invasomes, we aimed to produce liposomes by extrusion5 ofof 18 a homogenous solution comprising the respective terpene solved in ethanol, 0.9% saline and soybean lecithin.lecithin. ExtrusionExtrusion of of a a solution solution without without terpenoids terpenoids served served as as control control and and resulted resulted in thein the formation formation of invasomes,of invasomes, as visualizedas visualized by cryoby cryo transmission transmission electron electron microscopy microscopy (Cryo (Cryo TEM) TEM) (Figure (Figure2A). 2A).

FigureFigure 2.2. Successful production of of invasomes invasomes encapsulat encapsulatinging thymol, thymol, menthol, menthol, camphor camphor and and cineol cineol by byextrusion. extrusion. (A ()A Cryo) Cryo transmission transmission electron electron microscopy microscopy (TEM) (TEM) showing showing control invasomesinvasomes withoutwithout terpenoidsterpenoids (small(small representativerepresentative sectionsection ofof originaloriginal micrograph).micrograph). ((BB)) SchematicSchematic viewview ofof terpenoidsterpenoids encapsulatedencapsulated by by invasomes. invasomes. Localization Localization of of terpenoids terpenoids within within the the aqueous aqueous phase phase of of thethe invasomeinvasome was was chosenchosen onlyonly forfor visualizationvisualization reasonsreasons andand doesdoes not not represent represent their their natural natural localization. localization. (C(C––FF)) SmallSmall representativerepresentative sections sections of of original original cryo cryo electron electron micrographs micrographs revealed revealed invasomes invasomes comprising comprisingthymol thymol (C), menthol(C), menthol (D), camphor (D), camphor (E) or cineol (E) or (F )cineol after extrusion.(F) after TEM:extrusion. Cryo transmissionTEM: Cryo transmission electron microscopy. electron microscopy. We next applied the terpenoids thymol, menthol, camphor or cineol for production of invasomes (FigureWe2B). next Cryo applied TEM micrographsthe terpenoids showed thymol, the menthol, presence camphor of invasomes or cineol comprising for production thymol of (Figure invasomes2C), menthol(Figure 2B). (Figure Cryo2D), TEM camphor micrographs (Figure2 E)showed or cineol the (Figure presence2F) of after invasomes extrusion. comprising Interestingly, thymol Cryo (Figure TEM allowed2C), menthol us to observe(Figure multilamellar2D), camphor membrane (Figure 2E) boundaries or cineol (Figure in all five 2F) approaches after extrusion. (Figure Interestingly,2A,C–F). CryoDuring TEM allowed characterization us to observe of the multilamellar newly produced memb invasomesrane boundaries via Cryo in all TEM, five approaches we observed (Figure that encapsulation2A, C–F). of thymol and camphor resulted in a significant shift towards unilamellar vesicles. We suggestDuringthat characterization terpenoids such of asthe thymol newly might produced decrease invasomes membrane via fluidityCryo TEM, and thuswe observed lead to more that unilamellarencapsulation liposomes. of thymol On and the camphor contrary, resulted cineol-invasomes in a significant revealed shift towards a similar unilamellar distribution vesicles. between We unilamellarsuggest that and terpenoids bilamellar such vesicles as thymol comparable might decr to control.ease membrane Furthermore, fluidity cineol-invasomes and thus lead showedto more aunilamellar similar size liposomes. to control-invasomes, On the cont whilerary, encapsulationcineol-invasomes of thymolrevealed and a camphorsimilar distribution led to significantly between smallerunilamellar invasomes and bilamellar and menthol-comprising vesicles comparable invasomes to control. were Furthermore, significantly enlargedcineol-invasomes compared showed control. a similar size to control-invasomes, while encapsulation of thymol and camphor led to significantly smaller invasomes and menthol-comprising invasomes were significantly enlarged compared control. We suggest this observation to be based on the elevated water solubility of cineol (3500 mg/L) compared to camphor (1600 mg/L), thymol (900 mg/L) and menthol (420 mg/L). Analysis of Zeta potentials revealed approximately neutral potentials of control invasomes (−2 ± 5 mV, Figure 3A). We also observed approximately neutral Zeta potentials for invasomes encapsulating the terpenoids thymol (−3 ± 6 mV, Figure 3B), menthol (−1 ± 5 mV, Figure 3C), camphor (−2 ± 5 mV, Figure 3D) or cineol (0 ± 5 mV, Figure 3E).

Biomedicines 2020, 8, 105 6 of 18

We suggest this observation to be based on the elevated water solubility of cineol (3500 mg/L) compared to camphor (1600 mg/L), thymol (900 mg/L) and menthol (420 mg/L). Analysis of Zeta potentials revealed approximately neutral potentials of control invasomes ( 2 5 mV, Figure3A). We also observed approximately neutral Zeta potentials for invasomes − ± encapsulating the terpenoids thymol ( 3 6 mV, Figure3B), menthol ( 1 5 mV, Figure3C), camphor − ± − ± ( 2 5 mV, Figure3D) or cineol (0 5 mV, Figure3E). − Biomedicines± 2020, 8, x FOR PEER REVIEW± 6 of 18

Figure 3. Invasomes without tepenoids (A) and encapsulating the terpenoids thymol (B) menthol (C) Figure 3. Invasomes without tepenoids (A) and encapsulating the terpenoids thymol (B) menthol (C) camphor (D) or cineol (E) reveal neutral Zeta potentials. camphor (D) or cineol (E) reveal neutral Zeta potentials.

InIn terms terms of of invasomal invasomal stability, stability, high high Zeta Zeta potentialspotentials of at least ± 2020 mV mV are are generally generally considered considered as an indicator for electrostatical and steric stabilization of invasomes± [30,31]. Although all produced as an indicator for electrostatical and steric stabilization of invasomes [30,31]. Although all produced terpenoid-invasomes showed neutral Zeta potentials in the present study, we observed the presence terpenoid-invasomes showed neutral Zeta potentials in the present study, we observed the presence of of stable invasomes using Cryo TEM after extrusion. A limitation of our study was that the low values stable invasomes using Cryo TEM after extrusion. A limitation of our study was that the low values of zeta potential could only be measured with low precision, e.g., 3 mV ± 6 for thymol-containing of zeta potential could only be measured with low precision, e.g., 3 mV 6 for thymol-containing invasomes. Furthermore, measurements of Zeta potentials in nano carrier systems± such as invasomes invasomes.are hampered Furthermore, by measuring measurements limitations of arising Zeta potentials in diluted in samples. nano carrier Hence, systems plenty such of asparameters invasomes arewhich hampered influence by measuring zeta potentials limitations such arisingas viscosit in dilutedy, pH, samples.and dielectric Hence, constant plenty ofare parameters not correctly which influencereflected zeta in diluted potentials samples such as[32]. viscosity, Cryo TEM, pH, to and the dielectric best of our constant knowledge, are not does correctly not have reflected these in dilutedlimitations, samples since [32 samples]. Cryo TEM,with much to the higher best of concentrations our knowledge, of invasomes does not have could these be analysed limitations, in their since samplesnative withdiluent. much higher concentrations of invasomes could be analysed in their native diluent. InIn accordance accordance with with ourour findings, findings, Sebaaly Sebaaly and and colleagues colleagues reported reported neutral neutral Zeta potentials Zeta potentials of −3.9 of 3.9± 1.9 1.9mV mV for eugenol-loadedfor eugenol-loaded Lipoid Lipoid S100-liposomes S100-liposomes prepared prepared by ethanol by injection ethanol method. injection Although method. − ± Althoughthe authors the demonstrated authors demonstrated an increase an in increase liposome in size liposome and size size distribution and size distributionafter storage in after aqueous storage insuspension aqueous suspension at 4 °C for 2 at months, 4 ◦C for encapsulation 2 months, encapsulationefficiencies of eugenol efficiencies (86.6%) of were eugenol unchanged (86.6%) [33]. were We suggest that encapsulation efficiencies of our produced invasomes may not be affected over time, despite their neutral Zeta potentials. In addition to providing an increased bioavailability and a more controlled drug release, our approach may also facilitate topical administration of thymol-invasomes due to the high permeability rate of invasomes through the skin [2,21,24]. Notably, extrusion of solution without addition of ethanol for solving the terpenoid of interest did not result in the formation of invasomes (data not shown). In summary, we successfully prepared invasomes encapsulating the terpenoids thymol, menthol, camphor and cineol.

Biomedicines 2020, 8, 105 7 of 18 unchanged [33]. We suggest that encapsulation efficiencies of our produced invasomes may not be affected over time, despite their neutral Zeta potentials. In addition to providing an increased bioavailability and a more controlled drug release, our approach may also facilitate topical administration of thymol-invasomes due to the high permeability rate of invasomes through the skin [2,21,24]. Notably, extrusion of solution without addition of ethanol for solving the terpenoid of interest did not result in the formation of invasomes (data not shown). In summary, we successfully prepared invasomesBiomedicines encapsulating 2020, 8, x FOR PEER the terpenoidsREVIEW thymol, menthol, camphor and cineol. 7 of 18

3.2. Encapsulation3.2. Encapsulation of Terpenoids of Terpenoids Significantly Significantly Changes Changes LamellarityLamellarity and and Size Size of ofIn Invasomesvasomes without without Affecting Affecting BilayerBilayer Thickness Thickness We nextWe characterizednext characterized the producedthe produced invasomes invasomes in in more more detail detail in in terms terms of of their their lamellarity,lamellarity, size and bilayerand thickness. bilayer thickness. For investigation For investigation of lamellarity, of lamellar weity, determined we determined individual individual types types of lamellarof lamellar phase lipid bilayersphase lipid ranging bilayers from ranging one from lipid one bilayer lipid (MLV1)bilayer (MLV1) up to eight up to lamellareight lamellar phase phase lipid lipid bilayers bilayers (MLV8) (Figure(MLV8)4A). Cryo (Figure electron 4A). Cryo micrographs electron micrographs (see also Figure (see also2) served Figure for2) served determination for determination of the individual of the typesindividual of lamellar types phase of lamellar lipid bilayers, phase lipid which bilayers, we presentwhich we in present relation in relation to their to distribution. their distribution. Notably, we observedNotably,strong we observed differences strong in lamellaritydifferences ofin invasomeslamellarity dependingof invasomes on depending the added on terpenoids the added and in terpenoids and in comparison to control. Without the addition of a terpenoid (control, Figure 4B), comparison to control. Without the addition of a terpenoid (control, Figure4B), mostly unilamellar mostly unilamellar (37 ± 8%) and bilamellar vesicles (37 ± 13%) were formed (* p < 0.05, Figure 4B) (37 8%) and bilamellar vesicles (37 13%) were formed (* p < 0.05, Figure4B) and showed almost ± and showed almost equal proportions± (p = 0.4 was not considered significant, Mann–Whitney test, equalone-tailed). proportions (p = 0.4 was not considered significant, Mann–Whitney test, one-tailed).

FigureFigure 4. 4.Characterization Characterization of lamellarity lamellarity of ofthe theprod produceduced terpenoid-comprising terpenoid-comprising invasomes. invasomes. (A) (A) SchematicSchematic viewview ofof individualindividual types types of of lamellar lamellar ph phasease lipid lipid bilayers. bilayers. (B) (WiB)thout Without the addition the addition of a of a terpenoid (control, B), mostly unilamellar and bilamellar vesicles were formed in equal proportions. terpenoid (control, B), mostly unilamellar and bilamellar vesicles were formed in equal proportions. (C–E) Invasomes comprising of thymol or camphor showed mostly unilamellar vesicles and a (C–E) Invasomes comprising of thymol or camphor showed mostly unilamellar vesicles and a significantly smaller amount of bilamellar vesicles, while menthol-invasomes revealed no changes significantlybetween smallerMLV1 and amount MVL2. of(D) bilamellar Cineol-invasomes vesicles, revealed while a menthol-invasomes similar distribution between revealed unilamellar no changes betweenand MLV1bilamellar and vesicles. MVL2. (DistributionD) Cineol-invasomes of the individual revealed vesicle a similar types distributionis depicted in between relation unilamellarto their and bilamellarlamellarity vesicles.measured from Distribution respective of cryo the electron individual micrographs. vesicle types* p < 0.05 is was depicted considered in relation significant totheir lamellarity(Mann–Whitney measured test, from one-tailed). respective MLV: cryo Multilam electronellar micrographs. vesicles. (n.s. *meansp 0.05 not was significant) considered significant (Mann–Whitney test, one-tailed). MLV: Multilamellar vesicles. (n.s. means not significant) On the contrary, a shift towards 63 ± 11% unilamellar vesicles (MLV1, Figure 4C) and a Onsignificantly the contrary, decreased a shift towardsamount of 63 26 11%± 7% unilamellarbilamellar vesicles vesicles (MLV2, (MLV1, * Figurep < 0.05,4C) Figure anda 4C) significantly were ± decreasedobserved amount for invasomes of 26 7%comprising bilamellar of thymol. vesicles Prod (MLV2,uction of * p invasomes< 0.05, Figure with menthol4C) were resulted observed in a for significantly increased± amount of unilamellar vesicles (54 ± 18%) compared to MLV3–8 (* p < 0.05), but no significant changes between the amounts of MLV1 and MLV2 (p = 0.0571 was not considered significant, Mann–Whitney test, one-tailed, Figure 4D). Similarly to thymol-invasomes, encapsulation of camphor resulted in mostly unilamellar vesicles (59 ± 10%, * p < 0.05) and significantly decreased amounts of bilamellar vesicles (32 ± 12%, * p < 0.05, Figure 4E). Interestingly, invasomes containing cineol revealed a similar lamellarity as the control with a similar distribution between unilamellar (43 ± 3%) and bilamellar vesicles (45 ± 2%, p = 0.2000 was not considered

Biomedicines 2020, 8, 105 8 of 18 invasomes comprising of thymol. Production of invasomes with menthol resulted in a significantly increased amount of unilamellar vesicles (54 18%) compared to MLV3–8 (* p < 0.05), but no ± significant changes between the amounts of MLV1 and MLV2 (p = 0.0571 was not considered significant, Mann–Whitney test, one-tailed, Figure4D). Similarly to thymol-invasomes, encapsulation of camphor resulted in mostly unilamellar vesicles (59 10%, * p < 0.05) and significantly decreased amounts of ± bilamellar vesicles (32 12%, * p < 0.05, Figure4E). Interestingly, invasomes containing cineol revealed ± a similar lamellarity as the control with a similar distribution between unilamellar (43 3%) and Biomedicines 2020, 8, x FOR PEER REVIEW ± 8 of 18 bilamellar vesicles (45 2%, p = 0.2000 was not considered significant, Mann–Whitney test, one-tailed, ± Figuresignificant,4F). In addition,Mann–Whitney no MLV6–8 test, one-tailed, were observable Figure 4F). in invasomes In addition, encapsulating no MLV6–8 were cineol observable (Figure4F). in invasomesIn addition encapsulating to their lamellarity, cineol (Figure we measured 4F). and calculated the size of the produced invasomes (Figure5InA). addition All invasomes to their lamellarity, including thewe measured control showed and calculated a large distributionthe size of the in produced size but alsoinvasomes specific changes(Figure according 5A). All invasomes the encapsulated including terpenoid.the control showed Compared a large to distribution control invasomes in size but showing also specific a size distributionchanges according from about the 20 encapsulated up to 80 nm radius terpenoid. and aCompared mean of 40 to 15control nm (Figure invasomes5B), thymol-containing showing a size ± invasomesdistribution revealed from about a significantly 20 up to 80 smaller nm radius radius and of a 33 mean18 of nm 40 (Figure± 15 nm5C, (Figure *** p 5B),< 0.0001 thymol- was ± consideredcontaining significant, invasomes unpairedrevealed a t-test,significantly one-tailed). smaller Preparation radius of 33 ± of 18 invasomes nm (Figure with 5C, *** camphor p < 0.0001 also resultedwas considered in a significantly significant, smaller unpaired invasomes t-test, one-tailed). size (30 16 Preparation nm radius, of Figureinvasomes5E) compared with camphor to control also ± (***resultedp < 0.0001, in a unpairedsignificantly t-test, smaller one-tailed). invasomes On size the contrary,(30 ± 16 nm menthol-comprising radius, Figure 5E) compared invasomes to revealed control a (*** p < 0.0001, unpaired t-test, one-tailed). On the contrary, menthol-comprising invasomes revealed significantly increased radius of 58 22 nm (Figure5D) compared to all other terpenoid-encapsulating a significantly increased radius ±of 58 ± 22 nm (Figure 5D) compared to all other terpenoid- invasomes and control invasomes (*** p < 0.0001, unpaired t-test, one-tailed). Interestingly, although encapsulating invasomes and control invasomes (*** p < 0.0001, unpaired t-test, one-tailed). their distribution showed a peak at 35–40 nm radius, the size of invasomes with cineol (43 17 nm Interestingly, although their distribution showed a peak at 35–40 nm radius, the size of invasomes± radius, Figure5F) was similar to control (40 15 nm, p = 0.13 was not considered significant, unpaired with cineol (43 ± 17 nm radius, Figure 5F)± was similar to control (40 ± 15 nm, p = 0.13 was not t-test,considered one-tailed). significant, unpaired t-test, one-tailed).

FigureFigure 5. 5.Encapsulation Encapsulation ofof terpenoidsterpenoids directlydirectly aaffectsffects invasomes size. size. (A (A) )Example Example of ofa asmall small representativerepresentative section section of of an an original original cryo-electron cryo-electron micrograph used used for for measuring measuring invasomes’ invasomes’ size. size. (B)( ControlB) Control invasomes invasomes without without terpenoid terpenoid showing showing a mean a mean radius radius of 40 of15 40 nm. ± (15C) Thymol-comprisingnm. (C) Thymol- comprising invasomes revealed a smaller radius of 33 ± 18 nm compared± to control. (D) Production invasomes revealed a smaller radius of 33 18 nm compared to control. (D) Production of of invasomes with menthol resulted in an increased± invasome radius of 59 ± 22 nm. (E) Like thymol, invasomes with menthol resulted in an increased invasome radius of 59 22 nm. (E) Like thymol, camphor-invasomes also shower a smaller invasome size (30 ± 16 nm radius)± compared to control. (F) camphor-invasomes also shower a smaller invasome size (30 16 nm radius) compared to control. With a mean radius of 43 ± 17 nm, the size of invasomes with cineol± was similar to control. Frequency (F) With a mean radius of 43 17 nm, the size of invasomes with cineol was similar to control. Frequency plots of the radius distribution± of the invasomes. A fit with the Gaussian function is displayed as a plots of the radius distribution of the invasomes. A fit with the Gaussian function is displayed as a red line. red line. As a third parameter for characterization of our newly produced invasomes, thickness of the liposomal bilayer was measured by evaluating cryo-electron micrographs (Figure 6A). We observed no significant differences (Mann–Whitney test, one-tailed) in the liposomal bilayer thickness of control invasomes (4 ± 0.5 nm, Figure 6B) compared to menthol-invasomes (4 ± 0.3 nm, p = 0.2482, Figure 6D) and invasomes encapsulating camphor (4 ± 0.5 nm, p = 0.2987, Figure 6E). Invasomes produced with thymol revealed a slightly but significantly increased bilayer thickness of 5 ± 1 nm (Figure 6C) compared to control (** p < 0.01), camphor-invasomes (* p < 0.05) and cineol-invasomes

Biomedicines 2020, 8, 105 9 of 18

As a third parameter for characterization of our newly produced invasomes, thickness of the liposomal bilayer was measured by evaluating cryo-electron micrographs (Figure6A). We observed no significant differences (Mann–Whitney test, one-tailed) in the liposomal bilayer thickness of control invasomes (4 0.5 nm, Figure6B) compared to menthol-invasomes (4 0.3 nm, p = 0.2482, Figure6D) ± ± and invasomes encapsulating camphor (4 0.5 nm, p = 0.2987, Figure6E). Invasomes produced ± with thymol revealed a slightly but significantly increased bilayer thickness of 5 1 nm (Figure6C) Biomedicines 2020, 8, x FOR PEER REVIEW ± 9 of 18 compared to control (** p < 0.01), camphor-invasomes (* p < 0.05) and cineol-invasomes (** p < 0.01, Mann–Whitney(** p < 0.01, Mann–Whitney test, one-tailed). test, In contrast,one-tailed). cineol-comprising In contrast, cineol-comprising invasomes showed invasomes a minor showed decrease a in bilayerminor thickness decrease (4in bilayer0.5 nm, thickness Figure6 (4F) ± in 0.5 comparison nm, Figure to6F) the in comparison other approaches. to the other approaches. ±

FigureFigure 6. 6.Encapsulation Encapsulation of of terpenoids terpenoids does does notnot aaffectffect the bilayer thickness thickness of of invasomes. invasomes. (A ()A Example) Example of aof small a small representative representative section section of of an an original original cryo-electroncryo-electron microg micrographraph used used for for measurement measurement of ofthe the bilayer thickness. (B–F) A similar liposomal bilayer thickness was observable for control invasomes bilayer thickness. (B–F) A similar liposomal bilayer thickness was observable for control invasomes (4 ± 0.5 nm), thymol-invasomes (5 ± 1 nm) menthol-invasomes (4 ± 0.3 nm), camphor-invasomes (4 ± (4 0.5 nm), thymol-invasomes (5 1 nm) menthol-invasomes (4 0.3 nm), camphor-invasomes ±0.5 nm) or cineol-invasomes (4 ± 0±.5 nm). Frequency plots of the bi±layer thickness distribution of (4 0.5 nm) or cineol-invasomes (4 0.5 nm). Frequency plots of the bilayer thickness distribution of ±invasomes. A fit with the Gaussian± function is displayed as a red line. invasomes. A fit with the Gaussian function is displayed as a red line.

TakenTaken together, together, invasomes invasomes with with terpenoids terpenoids showedshowed a shift towards towards unilamellar unilamellar vesicles, vesicles, except except for cineol with a similar distribution between unilamellar and bilamellar vesicles comparable to for cineol with a similar distribution between unilamellar and bilamellar vesicles comparable to control. While the bilayer thickness of invasomes was comparable in all approaches, preparation of control. While the bilayer thickness of invasomes was comparable in all approaches, preparation of invasomes with thymol and camphor led to significantly smaller invasomes compared control. On invasomes with thymol and camphor led to significantly smaller invasomes compared control. On the the contrary, menthol-comprising invasomes were significantly enlarged and we observed the radius contrary, menthol-comprising invasomes were significantly enlarged and we observed the radius of of cineol-invasomes to be comparable to control. cineol-invasomes to be comparable to control. 3.3. Invasomes Encapsulating Thymol, Camphor and Cineol Show Bactericidal Activity against S. aureus in 3.3. Invasomes Encapsulating Thymol, Camphor and Cineol Show Bactericidal Activity against S. aureus in a a Growth-Inhibition Zone Assay Growth-Inhibition Zone Assay After successfully producing and characterizing terpenoid-comprising invasomes, we assessed After successfully producing and characterizing terpenoid-comprising invasomes, we assessed their potential bactericidal activity against S. aureus in a growth-inhibition zone assay. After exposure their potential bactericidal activity against S. aureus in a growth-inhibition zone assay. After exposure of S. aureus to different terpenoid-invasomes overnight, we determined the size of the inhibitory of S. aureus to different terpenoid-invasomes overnight, we determined the size of the inhibitory zones. Control invasomes without terpenoids did not result in growth inhibition of S. aureus (Figure zones.7A). Compared Control invasomes to unaffected without control, terpenoids we observed did a notclearly result visible in growthgrowth inhibition inhibition of of S.S. aureus aureus (Figureexposed7A). to Compared invasomes to comprising una ffected thymol, control, camphor we observed or cineol a clearly (Figure visible 7B,D,E). growth Interestingly, inhibition of S. aureusinvasomesexposed encapsulating to invasomes menthol comprising did not affect thymol, growth camphor of S. aureus or cineol (Figure (Figure 7C). 7StatisticalB,D,E). Interestingly, evaluation invasomesof the measured encapsulating areas of menthol inhibition did validated not affect a growthstrong and of S. significant aureus (Figure inhibition7C). Statisticalof bacterial evaluation growth by thymol-containing invasomes compared to all other terpenoid-invasomes and control (Figure 7F). However, invasomes comprising camphor or cineol still caused a significant increase in the zone of inhibition compared to control and menthol-invasomes, which revealed no zone of inhibition (Figure 7F).

Biomedicines 2020, 8, 105 10 of 18 of the measured areas of inhibition validated a strong and significant inhibition of bacterial growth by thymol-containing invasomes compared to all other terpenoid-invasomes and control (Figure7F). However, invasomes comprising camphor or cineol still caused a significant increase in the zone of inhibition compared to control and menthol-invasomes, which revealed no zone of inhibition (Figure7F). Biomedicines 2020, 8, x FOR PEER REVIEW 10 of 18

Figure 7. Invasomes encapsulating thymol, camphor and cineol show bactericidal activity against S. Figure 7. Invasomes encapsulating thymol, camphor and cineol show bactericidal activity against aureus in a growth-inhibition zone assay. (A) Control invasomes without terpenoids did not affect S. aureus in a growth-inhibition zone assay. (A) Control invasomes without terpenoids did not bacterial growth. (B) Strong growth inhibition of S. aureus exposed to invasomes comprising thymol. affect bacterial growth. (B) Strong growth inhibition of S. aureus exposed to invasomes comprising (C) Invasomes encapsulating menthol did not affect growth of S. aureus. (D–E) Growth inhibition of thymol. (C) Invasomes encapsulating menthol did not affect growth of S. aureus. (D–E) Growth S. aureus exposed to invasomes comprising camphor and cineol. (F) Statistical evaluation of the inhibition of S. aureus exposed to invasomes comprising camphor and cineol. (F) Statistical evaluation measured zones of inhibition validated the significant inhibition of S. aureus growth by thymol- of the measured zones of inhibition validated the significant inhibition of S. aureus growth by containing invasomes compared to all other terpenoid-invasomes and control. * p < 0.05 was thymol-containingconsidered significant invasomes (Mann–Whitney compared test, to allone-tailed). other terpenoid-invasomes (n.s. means not significant) and control. * p 0.05 was considered significant (Mann–Whitney test, one-tailed). (n.s. means not significant) In Table 1, the particle size of different terpenoid formulations is depicted as measured in Figure 5. InWhen Table the1, theformulations particle size are of sort di edfferent from terpenoid the highest formulations antibacterial is depictedactivity (thymol) as measured to the in lowest Figure 5. When(cineol), the formulations as measured in are Figure sorted 7, fromit becomes the highest eviden antibacterialt that the terpenoids activity with (thymol) the highest to the lowestantibacterial (cineol), asactivity measured have in the Figure highest7, it becomespolydispersity evident index that also the (Table terpenoids 1). with the highest antibacterial activity have the highest polydispersity index also (Table1). Table 1. Invasome Particle Size and the Polydispersity Index. Table 1. Invasome Particle Size and the Polydispersity Index. Formulation Particle Size (nm) Polydispersity Index FormulationThymol Particle 66 ± Size36 (nm) Polydispersity 0.3 ± 0.05 Index Camphor 60 ± 32 0.3 ± 0.04 Thymol 66 36 0.3 0.05 Cineol 86 ± ±34 0.2 ± 0.03± Camphor 60 32 0.3 0.04 Menthol 118 ±± 44 0.2 ± 0.03± Cineol 86 34 0.2 0.03 Control 80 ± ±30 0.1 ± 0.02± Menthol 118 44 0.2 0.03 ± ± Control 80 30 0.1 0.02 3.4. Flow Cytometric Analysis of Cell Death Validate± High Bactericidal Acti±vity of Thymol-Loaded Invasomes against Gram-Positive S. aureus To validate the strong bactericidal activity of thymol-invasomes against S. aureus in the growth- inhibition zone assay, we also assessed the anti-bacterial activity of thymol-invasomes quantitatively using flow cytometry. Bacterial cell death was measured by the DNA-intercalating dye Propidum Iodide (PI), which is incorporated only by dead cells. Prior to this analysis, we determined the encapsulation efficiency and loading capacity of thymol-invasomes to ensure proper encapsulation of the terpenoid. Here, we observed an encapsulation efficiency of 47 ± 13% as well as a loading

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3.4. Flow Cytometric Analysis of Cell Death Validate High Bactericidal Activity of Thymol-Loaded Invasomes against Gram-Positive S. aureus To validate the strong bactericidal activity of thymol-invasomes against S. aureus in the growth-inhibition zone assay, we also assessed the anti-bacterial activity of thymol-invasomes quantitatively using flow cytometry. Bacterial cell death was measured by the DNA-intercalating dye Propidum Iodide (PI), which is incorporated only by dead cells. Prior to this analysis, we determined the encapsulation efficiency and loading capacity of thymol-invasomes to ensure proper encapsulation of the terpenoid. Here, we observed an encapsulation efficiency of 47 13% as well as a loading ± capacityBiomedicines of 0.5 2020, 80.1%, x FOR for PEER invasomes REVIEW encapsulating thymol. Compared to untreated negative11 controlof 18 ± (4–9% cell death), 0.5 mg/mL thymol packaged in invasomes resulted in a profound cell death of 70%capacity (Figure of8 A).0.5 Exposure± 0.1% for ofinvasomes 1 mg /mL encapsulatin invasome-encapsulatedg thymol. Compared thymol to even untreated resulted negative in 75% control bacterial cell(4–9% death cell (Figure death),8B). 0.5Since mg/mL 2 mgthymol/mL packaged thymol packaged in invasomes in invasomes resulted in resulted a profound in onlycell death 9% PI-stained of 70% S. aureus(Figure(Figure 8A). Exposure8C), we of additionally 1 mg/mL invasome-encapsulated assessed the cell count thymol per second even resulted during in the 75% flow bacterial cytometric cell death (Figure 8B). Since 2 mg/mL thymol packaged in invasomes resulted in only 9% PI-stained S. measurement. Here, only around 1000 cells/second were observed in the S. aureus population aureus (Figure 8C), we additionally assessed the cell count per second during the flow cytometric treated with 2 mg/mL thymol-comprising invasomes, whereas the cell count for control conditions measurement. Here, only around 1000 cells/second were observed in the S. aureus population treated ranged around 40,000 cells/second (Figure8D). Treatment of S. aureus with 0.5 mg/mL or 1 mg/mL with 2 mg/mL thymol-comprising invasomes, whereas the cell count for control conditions ranged invasome-encapsulated thymol resulted in cell flow of around 33,000 cells/second (Figure8D). Thus, around 40,000 cells/second (Figure 8D). Treatment of S. aureus with 0.5 mg/mL or 1 mg/mL invasome- weencapsulated suggest that thymol 2 mg/mL resulted thymol in cell packaged flow of around in invasomes 33,000 cells/second already resulted (Figure in 8D). a nearly Thus, completewe suggest cell deaththat of2 mg/mLS. aureus thymolprior packaged to PI-staining in invasomes and following alreadyflow resulted cytometric in a nearly measurements. complete cell Wedeath conclude of S. invasomesaureus prior encapsulating to PI-staining thymol and following to be strongly flow cytometric bactericidal measurements. against Gram-positive We conclude bacteria invasomes such as S. aureusencapsulating. thymol to be strongly bactericidal against Gram-positive bacteria such as S. aureus.

FigureFigure 8. Flow8. Flow cytometric cytometric analysis analysis of cell of deathcell death validate validate invasomes invasomes encapsulating encapsulating thymol thymol to be stronglyto be bactericidalstrongly bactericidal against Gram-positive against Gram-positive bacteria like bacteriaS. aureus .(likeA ,BS.) Comparedaureus. (A,B to) untreatedCompared negative to untreated control, 0.5negative mg/mL control, or 1 mg 0.5/mL mg/mL thymol or packaged1 mg/mL thymol in invasomes packaged resulted in invasomes in a profound resulted in cell a profound death depicted cell bydeath PI-staining. depicted (C by) 2 PI-staining. mg/mL thymol (C) 2 packaged mg/mL thymol in invasomes packaged resulted in invasomes in only resulted 9% PI-stained in only S.9% aureus PI- . stained S. aureus. (D) Assessment of cell flow revealed only around 1000 cells/second in the S. aureus (D) Assessment of cell flow revealed only around 1000 cells/second in the S. aureus population treated population treated with 2 mg/mL thymol-comprising invasomes, suggesting a nearly complete cell with 2 mg/mL thymol-comprising invasomes, suggesting a nearly complete cell death prior to following death prior to following flow cytometric analysis. PI: Propidium iodide. flow cytometric analysis. PI: Propidium iodide. 3.5. Thymol-Loaded Invasomes Do Not Affect Survival of Gram-Negative E. coli Next to Gram-positive bacteria such as S. aureus, we assessed the potential anti-bacterial activity of thymol-invasomes against Gram-negative species such as E. coli. Notably, 0.5 mg/mL thymol packaged in invasomes resulted in only 0.2% PI-stained dead cells (Figure 9A). Treatment of E. coli with 1 mg/mL or 2 mg/mL invasome-encapsulated thymol only led to 6% or 5% cell death (Figure 9B,C). With regards to the low amount of PI-stained cells, we also assessed the cell count per second during the flow cytometric measurement. Here, we observed no relevant effects of the different concentrations of invasome-encapsulated thymol on the growth of E. coli (cell count per second, Figure 9D). In summary, the invasomes comprising thymol produced in this study are highly bactericidal to Gram-positive S. aureus, but do not affect survival of Gram-negative E. coli.

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3.5. Thymol-Loaded Invasomes Do Not Affect Survival of Gram-Negative E. coli Next to Gram-positive bacteria such as S. aureus, we assessed the potential anti-bacterial activity of thymol-invasomes against Gram-negative species such as E. coli. Notably, 0.5 mg/mL thymol packaged in invasomes resulted in only 0.2% PI-stained dead cells (Figure9A). Treatment of E. coli with 1 mg/mL or 2 mg/mL invasome-encapsulated thymol only led to 6% or 5% cell death (Figure9B,C). With regards to the low amount of PI-stained cells, we also assessed the cell count per second during the flow cytometric measurement. Here, we observed no relevant effects of the different concentrations of invasome-encapsulated thymol on the growth of E. coli (cell count per second, Figure9D). In summary, the invasomes comprising thymol produced in this study are highly bactericidal to Gram-positive S. aureus, but do not affect survival of Gram-negative E. coli. Biomedicines 2020, 8, x FOR PEER REVIEW 12 of 18

FigureFigure 9. Invasomes 9. Invasomes encapsulating encapsulating thymol thymol are arenot notbacteric bactericidalidal against against Gram-negative Gram-negative bacteria bacteria such such as as E.E. colicoli.(. A(A––CC)) ComparedCompared toto untreated untreated negative negative control, control, treatment treatment of ofE. E. coli coli. with. with 0.5 0.5 mg mg/mL,/mL, 1 mg 1 /mL or mg/mL2 mg /ormL 2 thymolmg/mL packagedthymol packaged in invasomes in invasomes did not resultdid not in elevatedresult in elevated amounts amounts of cell death. of cell (D death.) Assessment (D) Assessment of cell flow revealed no relevant effects of the different concentrations of invasome- of cell flow revealed no relevant effects of the different concentrations of invasome-encapsulated thymol encapsulated thymol on the growth of E. coli. on the growth of E. coli.

3.6.3.6. Growth-Inhibition Growth-Inhibition Zone Zone Assay Assay Shows Shows Strong Strong Bacteri Bactericidalcidal Activity Activity of Cineol of Cineol Invasomes Invasomes against against E. coli E. coli In additionIn addition to thymol-invasomes, to thymol-invasomes, we assessed we assessed the potential the potential bactericidal bactericidal activity activity of invasomes of invasomes encapsulatingencapsulating menthol, menthol, camphor camphor and andcineol cineol against against E. coli.E. In coli. lineIn with line our with flow our cytometric flow cytometric analysis analysis of ofcell cell death, death, thymol-inv thymol-invasomesasomes revealed revealed no elevated no elevatedgrowth inhibition growth of inhibition E. coli, similarly of E. colito control-, similarly to invasomes without encapsulated terpenoids (Figure 10A,B). While menthol-invasomes led to a slight control-invasomes without encapsulated terpenoids (Figure 10A,B). While menthol-invasomes led growth inhibition of E. coli (Figure 10C), invasomes encapsulating camphor showed no bactericidal to a slight growth inhibition of E. coli (Figure 10C), invasomes encapsulating camphor showed no activity against E. coli (Figure 10D). Notably, exposure of E. coli to invasomes loaded with cineol bactericidal activity against E. coli (Figure 10D). Notably, exposure of E. coli to invasomes loaded with resulted in a strong and significant growth inhibition (Figure 10E,F). cineol resulted in a strong and significant growth inhibition (Figure 10E,F).

Biomedicines 2020, 8, 105 13 of 18 Biomedicines 2020, 8, x FOR PEER REVIEW 13 of 18

Figure 10. 10. InvasomesInvasomes encapsulating encapsulating cineol cineol reveal reveal bactericidal bactericidal activity activity against against E. coli inE. acoli growth-in a inhibitiongrowth-inhibition zone assay. zone (A assay.,B) Control (A,B) Controlinvasomes invasomes without withoutterpenoids terpenoids or thymol or invasomes thymol invasomes did not affectdid not bacterial affect bacterial growth. growth.(C,D) While (C,D menthol-invasomes) While menthol-invasomes led to a slight led to inhibition a slight inhibition of E. coli ofgrowth,E. coli camphor-invasomesgrowth, camphor-invasomes did not affect did not growth affect of growth S. aureus. of S. (E aureus.) Strong(E growth) Strong inhibition growth inhibition of E. coli exposed of E. coli toexposed invasomes to invasomes comprising comprising cineol. cineol.(F) Statistical (F) Statistical evaluation evaluation of the of themeasured measured zones zones of of inhibition validated the the significant significant inhibition inhibition of of E.E. coli coli growthgrowth by by cineol-containing cineol-containing invasomes invasomes compared compared to all to otherall other terpenoid-invasomes terpenoid-invasomes and and control. control. * p *

We next determined the potential bactericidal acti activityvity of non-extruded terpenoids as a control to our encapsulation approach. In In contrast contrast to to terp terpenoidsenoids encapsulated in in invasomes invasomes (Figures (Figures 77–10),–10), application of non-extruded terpenoids did not result in growth inhibition of E. coli or S. aureus (data not shown). In In summary, summary, the the invasomes invasomes comprising comprising th thymolymol produced produced here here are are highly highly bactericidal bactericidal to Gram-positiveto Gram-positive S. aureus,S. aureus, whilewhile cineol-invasomes cineol-invasomes affect atheffect survival the survival of Gram-negative of Gram-negative E. coli (FiguresE. coli (Figures7–10). 7–10). Potential antibacterial mechanisms of invasome formulations with terpen terpenoidsoids are depicted in Figure 11.11. Although terpenoids such as limonene, cineole or beta-citronellene have been widely used for formulation of invasomes [26,27], they were mostly applied as permeation enhancer for transdermal delivery and bioavailability and not as bioactive components [2]. In the present study, we focused on the production of invasomes with terpenoids as the bioactive components to inhibit bacterial cell growth. We found our terpenoids-invasomes to be bactericidal against Gram-positive S. aureus, with increasing efficiency from cineol- and camphor-invasomes (moderate bactericidal activity) to thymol-invasomes showing the strongest bactericidal effects. These findings are in line with the commonly reported bactericidal activity of thymol, camphor and cineol [10,12,13]. Interestingly, Mulyaningsih and colleagues reported that exposure of MRSA even to high concentrations of cineol does not inhibit multi-resistant S. aureus. However, a combination of the terpene aromadendrene with cineol resulted in reduced bacterial cell growth [34]. Extending these findings, we show that encapsulation of cineol into invasomes alone is sufficient for inhibiting growth of S. aureus without the application of additional terpenes. In accordance to the strong bactericidal effects of thymol-invasomes observed here, encapsulation of thymol into other nanocarriers such as ethylcellulose/methylcellulose nanospheres

Biomedicines 2020, 8, 105 14 of 18 was also reported to preserve its anti-bacterial activity against S. aureus [35]. In contrast to invasomes encapsulating thymol, camphor and cineol, we observed no anti-bacterial effects for menthol-invasomes against S. aureus, which is contrary to the already described bactericidal activity of menthol [11,12]. With regard to the very low water-solubility of menthol, we suggest the invasomal packaging of menthol to be challenging, in turn, affecting its bactericidal activity. In this line, we observed an increased average size of menthol-invasomes compared to all other terpenoid-invasome preparations and control-invasomes lacking terpenoids. In addition polydispersity index as a measurement of the uniformity of invasome size distribution, with a higher value resulting in a broader distribution, was highest with thymol (0.3) and camphor (0.3), suggesting a correlation to antibacterial activity. Biomedicines 2020, 8, x FOR PEER REVIEW 14 of 18

Figure 11. Schematic view on invasomal packaging of thymol and its selective bactericidal activity Figure 11. Schematic view on invasomal packaging of thymol and its selective bactericidal activity against Gram-positive S. aureus. against Gram-positive S. aureus.

NextAlthough to Gram-positive terpenoids such bacterial as limonene, species cineole such or as beta-citronelleneS. aureus, we have also been investigated widely used potential for formulation of invasomes [26,27], they were mostly applied as permeation enhancer for transdermal anti-bacterial properties of our invasomes and particularly, thymol-invasomes on Gram-negative E. coli. delivery and bioavailability and not as bioactive components [2]. In the present study, we focused on Here, cineol-comprising invasomes led to a strong inhibition of E. coli growth, which is in line with our the production of invasomes with terpenoids as the bioactive components to inhibit bacterial cell previous observations [13]. In contrast to the strong bactericidal effects against S. aureus, cell growth growth. We found our terpenoids-invasomes to be bactericidal against Gram-positive S. aureus, with of Gram-negative E. coli was not affected by thymol-invasomes. These observations are contrary increasing efficiency from cineol- and camphor-invasomes (moderate bactericidal activity) to thymol- toinvasomes the findings showing by Salvia-Trujillo the strongest and bactericidal colleagues effects. reporting These afindings bactericidal are in activity line with of the essential commonly oils of thymereported (containing bactericidal thymol) activity after of incorporation thymol, camphor into nano-emulsionsand cineol [10,12,13]. [35]. Interestingly, In particular, Mulyaningsih nano-emulsions comprisingand colleagues essential reported oils ofthat thyme exposure with of heterogeneous MRSA even to dropletshigh concentrations sizes between of cineol 10 nm does to 500not nminhibit were shownmulti-resistant to reduce S. growth aureus. ofHowever,E. coli [ 36a combination]. However, of the the authors terpene appliedaromadendrene unfractionated with cineol essential resulted oils, suggestingin reduced a bacterial synergistic cell actiongrowthof [34]. many Extending terpenes these to findings, be necessary we show for that bactericidal encapsulation activity of cineol against Gram-negativeinto invasomes species. alone Interestingly,is sufficient Trombettafor inhibiting and growth coworkers of S. demonstrated aureus without that theS. aureusapplicationappears of to beadditional far more sensitiveterpenes. toIn thymolaccordance than toE. the coli strong[37], whichbactericidal is in line effects with of ourthymol-invasomes present data. The observed authors reportedhere, encapsulation a minimal inhibiting of thymol concentration into other ofnanocarriers 5.00 mg/mL such thymol as ethylcellulose/methylcellulose for E. coli [37], suggesting the concentrationnanospheres of was up also to 2 mgreported/mL thymol to preserve in the its invasomes anti-bacterial applied activity here against to be notS. aureus sufficient [35]. forIn contrast inhibition of toE. invasomes coli growth. encapsulating Furthermore, thymol,S. aureus camphoris known and cineol, to secrete we observed pore-forming no anti-bacterial toxins (PFTs), effects which for werementhol-invasomes shown to mediate against the release S. aureus of, encapsulatedwhich is contrary clove to oilthe fromalready liposomes. describedIn bactericidal particular, activity Cui and coworkersof menthol reported [11,12]. With that PFTsregard form to the pores very low within water-solubility the liposome of membranes, menthol, we suggest allowing the release invasomal of the encapsulatedpackaging of clove menthol oil and to be facilitating challenging, its antibacterial in turn, affecting activity. its bactericidal On the contrary, activity. liposomal In this line, packaged we cloveobserved oil had an noincreased bactericidal average eff sizeects of on menthol-invasomesE. coli, which does compared not secrete to all PFTs other and terpenoid-invasome thus prevents the preparations and control-invasomes lacking terpenoids. In addition polydispersity index as a release of antibacterial essential oil from the invasome [38]. Our present observations may suggest a measurement of the uniformity of invasome size distribution, with a higher value resulting in a similar mechanism for thymol-invasomes leading to its selective activity against S. aureus (Figure 11). broader distribution, was highest with thymol (0.3) and camphor (0.3), suggesting a correlation to antibacterial activity. Next to Gram-positive bacterial species such as S. aureus, we also investigated potential anti- bacterial properties of our invasomes and particularly, thymol-invasomes on Gram-negative E. coli. Here, cineol-comprising invasomes led to a strong inhibition of E. coli growth, which is in line with our previous observations [13]. In contrast to the strong bactericidal effects against S. aureus, cell growth of Gram-negative E. coli was not affected by thymol-invasomes. These observations are contrary to the findings by Salvia-Trujillo and colleagues reporting a bactericidal activity of essential oils of thyme (containing thymol) after incorporation into nano-emulsions [35]. In particular, nano- emulsions comprising essential oils of thyme with heterogeneous droplets sizes between 10 nm to 500 nm were shown to reduce growth of E. coli [36]. However, the authors applied unfractionated

Biomedicines 2020, 8, 105 15 of 18

In addition, electrophoretic mobility measurement revealed a harder surface of E. coli compared to S. aureus [39]. The softer surface of S. aureus mainly comprising peptidoglycan may facilitate entry of thymol-invasomes into the bacterial cells more easily compared to Gram-negative E. coli (Figure 11). Accordingly, we achieved a highly efficient killing of S. aureus with only 0.5 mg/mL thymol encapsulated in invasomes in the present study (Figure 11). In summary, we demonstrate the successful production of invasomes encapsulating thymol, menthol, camphor or cineol and show a strong selective activity of thymol-invasomes against Gram-positive S. aureus. As a further benefit of our approach, encapsulation of terpenoids into nanocarrier systems such as invasomes is suggested to increase stability and protect against environmental factors causing degradation [2,33,40]. Here, liposomes composed of lipoid S100 and cholesterol were reported to retain considerable concentrations of isoeugenol, pulegone, terpineol, and thymol liposomes even after 10 months [29]. The application of soy lecithin liposomes comprising cinnamon oil was further shown to improve stability of the essential oil and extend the bactericidal action time [20]. In addition, the application of invasomes was particularly found to elevate the stability of the encapsulated compounds (reviewed in [24]). There are several nanocarrier systems, encapsulating terpenoids, which are systematically reviewed in [2]. The formulation systems encapsulating terpenoids include polymer-based systems such as nano-capsules, nano-particles, nano-fibers and nano-gels. Furthermore, lipid-based systems are frequently used (67% of the formulations), presumably due to the low toxicity. A subgroup of lipid systems are the vesicular systems, which include invasomes. The most investigated biological activity of terpenoids in nano carrier systems is the anti-inflammatory action. Invasomes were used as anti-acne treatments, hypertension treatment and photosensation therapy [2]. Antimicrobial activity was reported with nano capsules with essential oils from lemon grass, nano emulsions with tea tree oil and penetration-enhancing vehicles with essential oil from Santolina insularis. Here, we report a novel antimicrobial application of invasome formulations with terpenoids. We conclude that our findings might provide a promising approach to increase the bioavailability of terpenoid-based drugs and might be applicable for treating severe bacterial infections such as MRSA in the future. In this regard, the major treatment aims of our formulations include a broad spectrum of applications, ranging from mucosal infections in airway diseases to systemic infections such as sepsis. In this direction, we have previously shown that patients with chronic rhinosinusitis have increased levels of S. aureus-containing biofilms in the nose [13]. Growth of S. aureus biofilms on the nasal mucosa could be inhibited by 1,8-cineol. Here, we extend these findings to thymol-containing invasomes, which have superior antibacterial activity than formulations with 1,8-cineol (see Figure7). Taken together, an invasome formulation as described here, containing thymol might be useful as an aerosol spray for pre-operative nose cleaning and might have fewer side effects in comparison to disinfectants directly applied on the mucosa. As a general use, it might be envisaged that invasomes containing thymol or other terpenoids could be employed to treat infected surfaces as in nose, lung and skin wounds. Finally, invasomes containing terpenoids might be used in addition or as an alternative to antibiotics.

Author Contributions: Conceptualization, A.H., C.K. and B.K.; validation, A.H., C.K. and B.K.; formal analysis, B.P.K., J.F.W.G., I.E. and A.H.; investigation, B.P.K., I.E., J.F.W.G. and R.D.; resources, A.H., C.K., B.K. and A.P.; data curation, A.H., C.K., B.K. and J.F.W.G.; writing—original draft preparation, B.P.K. and J.F.W.G.; writing—review and editing, A.H., C.K., B.K., I.E., R.D. and A.P.; visualization, B.P.K. and J.F.W.G.; supervision, A.H., C.K. and B.K.; project administration, A.H., C.K. and B.K.; funding acquisition, A.H., C.K., B.K. and A.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University of Bielefeld and received no external funding. Acknowledgments: The excellent technical help of Angela Kralemann-Köhler is gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Biomedicines 2020, 8, 105 16 of 18

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