Chemistry and Physics of Lipids 243 (2022) 105174

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Chemistry and Physics of Lipids

journal homepage: www.elsevier.com/locate/chemphyslip

Resveratrol loaded in cationic glucosylated liposomes to treat

Staphylococcus epidermidis infections
Livia Pagano a, Foteini Gkartziou b, Stefano Aiello a, Beatrice Simonis a, c, Francesca Ceccacci c,
Simona Sennato d, Alessia Ciogli e, Spyridon Mourtas f, Iris Spiliopoulou g,
Sophia G. Antimisiaris b, f, Cecilia Bombelli c, *, Giovanna Mancini h
a
Department of Chemistry, Sapienza University, P.Le A. Moro 5, Rome, Italy
b
Institute of Chemical Engineering Sciences, FORTH/ICE-HT, Platani, 26504 Patras, Greece
c
CNR-Institute for Biological Systems (ISB), Secondary Office of Rome-Reaction Mechanisms c/o Department of Chemistry, Sapienza University, P.le A. Moro 5, Rome,
Italy
d
CNR-Institute of Complex Systems (ISC)-Sede Sapienza c/o Physics Department, Sapienza University, P.le A. Moro 5, Rome, Italy
e
Department of Chemistry and Technology of Drug, Sapienza University, P.le A. Moro 5, Rome, Italy
f
Lab Pharm Technology, Department of Pharmacy, School of Health Sciences, University of Patras, 26504 Rio-Patras, Greece
g
Department of Microbiology, School of Medicine, University of Patras, Rio, 26504 Patras, Greece
h
CNR-Institute for Biological Systems (ISB), Area della Ricerca di Roma 1, Via Salaria Km 29,300, 00015 Monterotondo, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: Glucosylated liposomes composed of the natural saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phos­

Cationic liposomes phocholine (DPPC), cholesterol (Chol) and a cationic amphiphile featuring a glucosyl moiety (GL4), have been
Glucosylated liposomes developed for delivering the antimicrobial trans-Resveratrol (RSV) to S. epidermidis, characterized by
Glucosylamphiphile
carbohydrate-specific adhesins able to recognize glucose. The cationic derivative of cholesterol, DC-Chol, was
trans-Resveratrol
Staphylococcus epidermidis infections
also included in liposome formulations, alone or in combination with GL4, in order to explore the role of both
Sonication vs extrusion cationic charge and sugar moiety in the interaction of liposomes with bacterial cells. RSV was included inside
glucosylated cationic liposomes by the thin film method, coupled with either extrusion or sonication; liposome
mean diameter, polydispersity index, surface charge, RSV entrapment efficiency and concentration have been
measured by DLS, electrophoretic mobility, and HPLC. The antimicrobial activity of RSV-loaded liposomes was
evaluated by monitoring the bacterial growth curves of two cell lines of Staphylococcus epidermidis, a slime
positive strain (i.e. a strain able to form a biofilm) and a slime negative one. Results point out that, when the
glucosylamphiphile GL4 is included in the formulation, only the extrusion protocol allows obtaining mono­
disperse liposomes with high RSV entrapment efficiency. The mean diameters of empty and resveratrol-loaded
liposomes are all around 120–140 nm and size distribution are narrow, except for samples including GL4 at 5
molar percentage. Here the higher polydispersity index may be the indication of the occurrence of a restructuring
phenomenon. The microbiological tests put in evidence a different response of the two bacterial cell lines to
liposome treatments, in fact, the slime negative bacterial cells, that are not able to produce the extracellular
polymeric substances, are more susceptible to the cationic charge of the liposomes and to the detergent effect of
GL4. The most interesting results concern DPPC/Chol/GL4 liposomes on the slime positive strain: this formu­
lation, non-toxic in itself, displays an enhanced antibacterial efficacy with respect to free RSV, killing bacteria
even at concentration tenfold under the MIC.

1. Introduction organism living on our skin, turned out to be an opportunistic pathogen

(Christensen et al., 1982) that is often associated to nosocomial and
Staphylococcus epidermidis, for many years considered a harmless device-related infections (Götz, 2002; Cerca et al., 2011).

* Correspondence to: CNR-Institute for Biological Systems (ISB), Secondary Office of Rome-Reaction Mechanisms c/o Department of Chemistry, Sapienza Uni­
versity, P.le A. Moro 5, 00185 Rome, Italy.
E-mail address: cecilia.bombelli@cnr.it (C. Bombelli).

https://doi.org/10.1016/j.chemphyslip.2022.105174
Received 7 October 2021; Received in revised form 17 December 2021; Accepted 11 January 2022
Available online 14 January 2022
0009-3084/© 2022 Elsevier B.V. All rights reserved.
L. Pagano et al.
Chart 1. Molecular structures of t-Resveratrol and of liposome components.
In contrast with S. aureus, it has a relatively small amount of toxins
able to cause damages on tissues and for this reason it is often cause of
subacute or chronic infections (Vuong and Otto, 2002).
A wide variety of drugs has been used for the treatment of
S. epidermidis infections, but the slime capsule of Staphylococci acts like a
natural barrier for many antibiotics, and bacterial drug resistance is now
a major issue. In the 90s it was estimated that up to 80% of the
S. epidermidis strains were resistant to methicillin (Rupp and Archer,
1994; Diekema et al., 2001). S. epidermidis strains have acquired resis­
tance to several other antibiotics, including rifamycin, fluoroquinolones,
gentamicin, tetracycline, chloramphenicol, erythromycin, clindamycin,
and sulfonamides (Rogers et al., 2009). In addition, several resistance
genes have been shown to be transferred from S. epidermidis to other
species, S. aureus among others (Forbes and Schaberg, 1983; Espinosa
et al., 2017). For all these reasons, the use of drugs not triggering drug
resistance should be explored and encouraged in order to safely and
efficiently treat subacute and chronical infections caused by
S. epidermidis. The search for non-antibiotic strategies has renewed the
interest towards the world of natural bioactive compounds that could
exploit alternative mechanisms to fight bacteria, alone or in combina­
tion with antibiotics (Khameneh et al., 2019). Among the natural sub­
stances attracting research attention, there is Resveratrol (3,5,
4’-trihydroxystilbene, RSV), an antimicrobic/antioxidant phenolic
compound produced by many plants when attacked by bacteria, fungi,
insects or even UV rays. Its first appearance in modern science is dated
back to 1939, when Takaoka first isolated it from the roots of Veratrum
grandiflorum (Takaoka, 1939).
RSV possesses a wide spectrum of antimicrobial activity, showing
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Chemistry and Physics of Lipids 243 (2022) 105174
antimicrobial effect on several bacterial strains, namely Staphylococcus
aureus, Bacillus cereus, S. epidermidis (Gutiérrez et al., 2017; Kolouchová
et al., 2018; Chan, 2002). Moreover, biological experiments on many
bacteria, among which S. epidermidis, showed that RSV can be also useful
to prevent biofilm growth (Takaoka, 1939; Gutiérrez et al., 2017; Chan,
2002), as RSV is able to inhibit the process of Quorum Sensing (QS)
among bacterial cells by blocking their initial adhesion to the surface
(Paulo et al., 2010).
Thanks to these features, to its safety profile and relatively low cost,
this molecule has drawn the attention of pharmaceutical research.
However, the use of RSV is hampered by its low solubility in aqueous
media and instability when exposed to light, as well as by its low
specificity for bacteria, all issues that could be solved by developing a
suitable drug delivery system. Indeed, in the last ten years, there has
been a growing interest in the use of nanocarriers, and in particular li­
posomes, to develop optimal drug delivery systems against bacterial
biofilm.
The inclusion of RSV into cationic liposomes enhances its water
solubility and stability, prevents its degradation (Jøraholmen et al.,
2015), and favors the interaction with bacteria biofilms (Jøraholmen
et al., 2015; Kim and Jones, 2004; Kim et al., 1999). Moreover, the
insertion of glycosylated moieties on the external surface of liposomes
can promote the targeting of liposomes towards bacterial cells and
biofilms over-expressing carbohydrate-specific adhesins (i.e. lectins)
(Ofek et al., 2003).
Here we report on the loading of RSV inside glucosylated cationic
liposomes following two different liposome preparation techniques
(sonication and extrusion), starting from a thin lipid film including RSV.
L. Pagano et al.
Biological assays were carried out on the most suitable liposome for­
mulations to evaluate the antimicrobial activity of RSV-loaded lipo­
somes against S. epidermidis.
The investigated liposome formulations are composed of 1,2-dipal­
mitoyl-sn-glycero-3-phosphocholine (DPPC, a saturated phospholipid),
cholesterol (Chol), 3ß-[N-(N’,N’-dimetyl-aminoethane)-carbamoyl]
cholesterol (DC-Chol) and GL4, a cationic glucosylated lipid (Chart 1)
previously synthesized and used in our laboratory to target bacteria and
biofilms (Mauceri et al., 2014; Francolini et al., 2019; Aiello et al.,
2021). As previously reported, DOPC based liposomes including GL4 are
able to improve the antibiofilm activity of usnic acid towards S.epi­
dermidis (Francolini et al., 2019), and, most importantly, in these lipo­
somes the presence of both a cationic function and a glucosyl moiety is
critical for the efficiency of delivery (Francolini et al., 2019).
The cationic lipid DC-Chol is used in order to confer a higher cationic
charge on liposomes; its presence in the formulation resulted in higher
efficiency compared to other cationic amphiphiles when used in lipo­
somes tailored to target bacteria (Kim and Jones, 2004; Kim et al., 1999;
Jones et al., 1997; Robinson et al., 2001).
In the present work, neutral liposomes only containing DPPC or
DPPC/Chol as well as cationic liposomes containing DPPC, Cholesterol
and DC-Chol in the absence of a glycosylated lipid were also investigated
to better understand the role of both cationic charge and sugar moieties
in the interaction of liposomes with bacterial cells.
The size (Dh), size distribution (PDI) and the ζ-potential of empty and
RSV-loaded liposomes were evaluated by Dynamic Light Scattering
(DLS) and Laser Doppler Electrophoresis (LDE) measurements, while
RSV entrapment efficiency was assessed by HPLC analysis.
The antimicrobial activity of these RSV-loaded liposome formula­
tions was then evaluated on two cell lines of S. epidermidis, a slime
positive strain (i.e. a strain able to produce the Extracellular Polymeric
Substances-EPS) and a slime negative strain, and compared to that of
free RSV.
2. Materials and methods
2.1. Materials
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 3ß-[N-(N’,
N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-
Chol) were purchased from Avanti Polar Lipids (Alabaster, AL, USA);
phosphate-buffered saline (PBS; 0.01 M phosphate buffer; 0.0027 M
KCl; 0.137 M NaCl; pH 7.4), trans-Resveratrol (purity = 99%), trans-
Stilbene (purity = 96%), cholesterol (purity = 99%), cellulose dialysis
membrane (D9652–100FT, m.w. cut off = 14,000 Da), TSB (Tryptic soy
agar), TSA (Tryptic soy agar broth), Crystal Violet (Tris(4-(dimethyla­
mino)phenyl)methylium chloride, purity ≥ 90.0%), ethanol
(purity ≥ 95.0%), isopropyl alcohol (purity ≥ 99.7%),dimethox­
ydimethylsilane, were purchased from Sigma Aldrich.
To activate dialysis membrane the tubing was: 1) washed in running
water for 3–4 h; 2) treated with a 0.3% (w/v) solution of sodium sulfide
at 80 ◦ C for 1 min; 3) washed with hot water (60 ◦ C) for 2 min, followed
by acidification with a 0.2% (v/v) solution of sulfuric acid, 4) rinsed
with hot water to remove the acid.
GL4 was synthesized as previously described (Mauceri et al., 2015).
2.2. Preparation of empty and resveratrol-loaded liposomes
Mixed liposomes were prepared according to the thin film hydration
method coupled with a freeze-thaw protocol, followed by extrusion or
sonication. In brief, the proper amounts of lipids (DPPC, Chol, DC-Chol
and GL4) were dissolved in CHCl3 and mixed in a round bottom flask.
For RSV-loaded formulations, the proper amount of a RSV solution in
absolute ethanol was added in order to have a 1: 8 RSV/lipids ratio. The
solvent was removed by rotary evaporation (Buchi Rotavapor R-210;
Buchi Vacuum controller V-800 R-200) to obtain the formation of a thin
3
Chemistry and Physics of Lipids 243 (2022) 105174
lipid film on the bottom wall of the flask. The lipid film was then dried
under high vacuum overnight to remove traces of organic solvents, and
then hydrated with phosphate buffered saline (PBS) solution to give a
10, 20 or 40 mM liposomal suspension.
The aqueous suspension was vortex-mixed in order to completely
detach the film from the flask and the obtained multilamellar vesicles
(MLVs) were freeze-thawed six times from liquid nitrogen to 50 ◦ C.
Samples containing RSV were sonicated (probe tip sonicator Sonics
Vibra-Cell) at 40 W (10 cycles of 10 s) in order to improve entrapment
inside the lipid bilayer by breaking RSV aggregates that usually form in
aqueous solutions (Bonechi et al., 2012).
MLVs were then extruded or sonicated to produce SUVs, as described
in the following.
2.2.1. Extrusion protocol
MLVs were extruded 10 times through a 100 nm polycarbonate
membrane (Whatman Nuclepore) at temperature higher than Tm, and
under high pressure (up to 40 atmospheres) using a 10 mL extruder
(Lipex Biomembranes). The removal of unentrapped RSV was performed
by dialysis (membrane molecular weight cut-off: 14.000 kDa) following
a protocol previously described (Aiello et al., 2021): the internal volume
inside the tubing, containing the liposome formulation, was dialyzed
versus a 25-times larger external PBS volume, and the external PBS
changed four times over 2.5 h. Total lipid concentration in liposome
dispersions before and after dialysis was determined by 1HNMR (NMR
Bruker Avance 300) in the presence of an internal standard, dimethox­
ydimethylsilane, following a protocol previously described (Bombelli
et al., 2005).
2.2.2. Sonication protocol
MLVs were sonicated at 225 Watt for 1 h (probe tip sonicator Sonics
Vibra-Cell). To avoid overheating of lipid suspension, test tube con­
taining the sample was placed in a water bath. Liposomal formulations
were centrifuged at 12.000 g (Centrifuge SciLogex D2012 Plus) for
15 min to remove precipitated lipids and titanium particles released by
the sonication tip. Unentrapped RSV was removed by centrifugation at
20.000 g at 4 ◦ C for 1 h (Thermo Scientific Sorvall WX Ultra Series Ul­
tracentrifuge). The supernatant was discarded, precipitated SUVs were
resuspended in PBS (the same volume added for film hydration) and
placed at 50 ◦ C for the annealing process. The Stewart assay was carried
out to quantitate the phospholipid concentration of liposome disper­
sions (Stewart, 1980).
2.3. Characterization of liposomes
Suspensions of empty and RSV-loaded liposomes, prepared both by
extrusion and sonication protocol, were properly diluted (1 mM total
lipids) in 150 mM PBS and analyzed by Dynamic Light Scattering (DLS)
and Laser Doppler Electrophoresis (LDE) soon after preparation to
determine their average diameter, polydispersity index and ζ-potential.
A Malvern Nano-ZetaSizer apparatus, operating in backscattering
(173◦ ) and equipped with a 5 mW HeNe laser (λ = 632.8 nm), a ther­
mostatted sample holder and a digital logarithmic correlator was used to
perform both DLS and LDE measurements. Both size and ζ-potential
were measured at 25 ◦ C.
DLS autocorrelation functions were analyzed by using the cumulant
method (Koppel, 1972). The first cumulant was used to obtain the
apparent diffusion coefficients D of the particles, further converted into
apparent hydrodynamic diameters, Dh, by using the Stokes–Einstein
relationship Dh = kBT/3πηD, where kBT is the thermal energy and η is
the solvent viscosity. For samples with PDI larger than 0.3, we have
performed a size distribution analysis by NNLS algorithm in order to
ascertain the presence of different population in the samples.
In LDE measurements, phase analysis light scattering (PALS;
Tscharnuter, 2001) was employed to analyze the Doppler shift and
obtain the ζ-potential. Liposome used in these experiments were diluted
L. Pagano et al. Chemistry and Physics of Lipids 243 (2022) 105174

Table 1 TS Broth in the wells. Two plates were used for each assay, one for the
Liposome formulations prepared by the extrusion protocol and evaluated on free RSV and one for liposome samples.
S. epidermidis. Total lipid concentration is 20 mM and RSV/lipids molar ratio at
the beginning of the preparation is 1:8. In the last column is reported the con­ 2.5.2. Growth curves
centration of RSV in the final formulations, assed by HPLC. Error associated to The antimicrobial activity was evaluated by monitoring the bacterial
RSV concentration values is the standard deviation of three repeated measure­
growth curves in the presence of 0.07, 0.14, 0.35 and 0.70 mM RSV
ments on three different samples.
(MIC50 =0.6 mM) (Kolouchová et al., 2018), either free or loaded in
Formulation Lipid ratio [RSV] (mM) liposomes. Empty liposomes were also evaluated for comparison.
LPs and GLPs Growth curve were obtained measuring the optical density (O.D.) at
DPPC 100 – 590 nm (UV–VIS spectrophotometer (Shimadzu Mini 1240)) of liquid
DPPC/RSV 100 2.40 ± 0.05
cultures incubated at 37 ◦ C every 2 h until bacteria growth reached a
DPPC/Chol 80:20 –
DPPC/Chol/RSV 80:20 1.00 ± 0.02 plateau (stationary phase). This technique is based on the correlation
DPPC/Chol/GL4 75:20:5 between turbidity of bacterial solution and the number of bacterial cells
DPPC/Chol/GL4/RSV 75:20:5 2.00 ± 0.05 present in the solution. Liposomes themselves cause turbidity to solu­
DC-Chol-LPs and DC-Chol-GLPs tions. Thus, for the subtraction of background turbidity generated by
DPPC/Chol/DC-Chol 60:10:30
liposomes, six wells per liposome sample were loaded only with lipo­
DPPC/Chol/DC-Chol/RSV 60:10:30 1.40 ± 0.01
DPPC/Chol/DC-Chol/GL4 60:10:20:10 somes and broth and were measured throughout the experiments for the
DPPC/Chol/DC-Chol/GL4/RSV 60:10:20:10 2.00 ± 0.03 subtraction of background turbidity generated by liposomes. The O.D.
media of these blanks was subtracted to the appropriate O.D. media
measured in the presence of bacteria. Some wells have been loaded only
up to 1 mM -with PBS buffer and low applied voltages were used to with broth and measured throughout the experiments in order to
avoid the risk of effects due to Joule heating. exclude any possibility of contamination. These O.D values were stable
throughout the experiment, indicating that no bacterial contamination
2.4. Determination of trans-resveratrol entrapment efficiency took place. The values of the blanks were subtracted from the corre­
sponding sample O.D values measured in the experiments.
The extent of RSV enclosed in liposomes was assessed by HPLC
analysis (Waters Alliance 2695) coupled with a photodiode array de­ 2.6. Statistical evaluation
tector (PDA Waters 996). Data were collected and analyzed using the
Empower 2 software (Waters, Milford, MA, USA). The column used for Statistical significance for all comparisons was set at p = 0.05. Data
the analysis was a C18-SunFire 150 × 4.6 mm ID, 3.5 µm (dp). The were analyzed by using two-way ANOVA followed by Tukey’s post hoc
mobile phases were water/acetonitrile 95/5 + 0.1% TFA (solvent A) test. The significance of comparisons is presented on the graphs and/or
and methanol + 0.1% TFA (solvent B). Both solvents were filtered mentioned in discussion.
through 0.45 µm filters before use. The analysis was carried out at 30 ◦ C
at 1 mL/min flow rate. Trans-Stilbene (TSB) was used as internal stan­ 3. Results and discussion
dard (concentration 1 × 10-4 M). A calibration curve with resveratrol
standard solutions in absolute ethanol was built in the concentration With the aim of developing liposome formulations able to specif­
range 8.1 × 10-5–8.1 × 10-4 M. ically deliver antimicrobial trans-RSV to S. epidermidis, we prepared
Before injection, liposomal samples were diluted with a methanol glucosylated liposomes including in their formulation a cationic gluco­
solution containing TSB (1 × 10-4 M final concentration). Dilution in sylated amphiphile (GL4), a saturated phospholipid (DPPC), cholesterol
methanol is needed to break liposomes and likely RSV clusters present in (Chol) and the cationic derivative of cholesterol DC-Chol. Non-gluco­
the samples, thus achieving a reliable evaluation of RSV content. sylated liposomes, both neutral (DPPC and DPPC/Chol) and cationic
The entrapment efficiency (EE%) was then evaluated by the (DPPC/Chol/DC-Chol), either empty or loaded with RSV were also
following equation: prepared for comparison (Table 1). DC-Chol displays several functions,
such as efficiently interacting with bacteria and stabilizing lipid bilayers
[L]0 [RSV]pd
EE(%) = × 100 in the presence of other amphiphiles. In this study DC-Chol was
[RSV]0 [L]pd
particularly useful because it also allowed investigating independently
the role of both cationic surface charge and glucose moiety on the sur­
where [RSV]pd and [L]pd indicate, respectively, the concentration of RSV
face of liposomes. Cholesterol was added to all the formulations to make
and total lipids after dialysis and [RSV]0and [L]0 those after extrusion or
lipid bilayer more stable, thus allowing the addition of a large amount of
after sonication.
GL4. Indeed GL4 has detergent properties and, depending on its per­
centage and overall concentration, it can destabilize the lipid bilayer in
2.5. Microbiological test the absence of cholesterol, leading to the formation of micellar aggre­
gates (Mauceri et al., 2014).
RSV-loaded liposome formulations reported in Table 1 were evalu­ We first explored the best preparation protocol to obtain mono­
ated for their antimicrobial activity with respect to free RSV against two disperse liposomes with the highest RSV entrapment efficiency. To this
strains of S. epidermidis, a slime negative strain (ATCC 12228) and a aim, we formulated mixed liposomes with DPPC, Chol and GL4 at two
slime positive one (ATCC 35984). different total lipid concentrations, 10 and 40 mM (see Table S1 in the
SI). Liposomes were prepared by the thin film method coupled with the
2.5.1. Bacterial strains culture freeze-thaw procedure, followed by either extrusion or sonication; in
Bacteria were first inoculated on Petri dish with Trypticase Soy Agar both protocols, samples containing RSV were submitted to additional
(TSA) and incubated overnight at 37 ◦ C. The day after, 4–5 colonies sonication cycles before freeze-thaw to break RSV aggregates that usu­
were added to 2 mL of Trypticase Soy Broth (TS Broth) and grown ally form in aqueous solutions and increase its entrapment in the lipid
overnight at 37 ◦ C after vortexing the solution. These inocula were used bilayer (Bonechi et al., 2012). Free RSV was removed by dialysis from
for the following microbiological tests. All the assays were performed extruded liposomes and by ultracentrifugation from sonicated ones.
using a sterile 96 wells micro-titration plate (400 μl well total volume). Entrapment efficiency (EE%) of RSV was evaluated by HPLC
The third day, 2 μl of bacteria liquid subculture were added to 150 μl of measurements.

4
L. Pagano et al.
The extrusion protocol turns out to be preferable as it provides li­
posomes with higher EE% of RSV and monodisperse vesicles in the
presence of the glucosylated derivative GL4. These results (reported in
Table S1 of the SI) are in agreement with data reported in the literature,
concerning the inclusion of RSV in neutral liposomes. (Isailović et al.,
2013).
Based on these results, the preparation of liposomes for microbio­
logical experiments was carried out only by the extrusion protocol.
Physicochemical features of the formulations used in the experiments on
S. epidermidis are reported in Fig. 1.
In a previous work, we investigated similar liposomes for the de­
livery of RSV, comprising both cholesterol and the glycosylated
amphiphile GL4 but the unsaturated phospholipid DOPC was used in
place of DPPC (Aiello et al., 2021). As might be expected, the nature of
the hydrophobic chains has a strong influence on the organization of the
bilayer as a whole and, as a consequence, on some physico-chemical
features of liposomes. In particular, while RSV EE% is more or less the
same independently of the phospholipid, the charge and the diameter of
the vesicles change differently in the two systems upon RSV and GL4
addition. The organization of mixed liposomes composed of GL4 and a
phosphocholine depends on different parameters such as temperature,
phase state of the membrane, and lipid chains (grade of unsaturation
and length) (Villalva et al., 2017). The different organization affects, in
turn, the packing of the mixed bilayer, the exposure of the positive
charge and the sugar moiety and, consequently, the physico-chemical
parameters of liposomes.
5
Chemistry and Physics of Lipids 243 (2022) 105174
Fig. 1. Physico-chemical properties of 20 mM
liposomes (RSV/lipids ratio at the beginning of
the preparation is 1:8) prepared by the extru­
sion protocol and evaluated on S. epidermidis.
Dh and PDI are the hydrodynamic diameter and
the polydispersity index, respectively, calcu­
lated by the cumulants method; EE% is the
entrapment efficiency determined by the ratio
of RSV concentration after dialysis and after
extrusion, assessed by HPLC measurements.
Solid bars correspond to RSV-loaded liposomes,
stripped bars to the empty ones. Error bar
associated to Dh, PDI, ζ-pot and RSV concen­
tration is the standard deviation of three
repeated measurements on three different
samples.*For these samples cumulant analysis
was compared with distribution analysis by
NNLS algorithm (see text).
The mean diameter of empty and RSV-loaded liposomes composed of
DPPC, Chol and GL4 are all around 120–130 nm, in agreement with the
dimension imposed by the extrusion membrane (100 nm). Size distri­
bution is relatively narrow (PDI 0.1 – 0.2), except for samples including
GL4 at 5 molar percentage, which have a higher Dh and a larger PDI. For
empty DPPC/Chol/GL4 liposomes, NNLS analysis evidences the pres­
ence of two distinct populations, with mean size 218 nm and 510 nm,
where the smallest one is the most abundant and in qualitative agree­
ment with the size determined by cumulant (around 95% according to
number ratio, obtained by NNLS- number weighted analysis). Also in the
presence of RSV, NNLS analysis evidences the presence of two distinct
populations, with mean size 278 nm and 650 nm, where the smallest
one is the most abundant (around 95%). The here observed growth of
vesicles is a general phenomenon, detected in many phospholipid/sur­
factant systems. At GL4 concentrations just below that inducing the
breaking of vesicle (with consequent formation of open lamellar struc­
tures) the amphiphile is able to induce fusion between the vesicles, and
formation of intermediate disks that evolve very rapidly in new vesicles,
with a diameter that is controlled by GL4concentration (Edwards et al.,
1993).
Liposomes containing DPPC, Chol, GL4 and DC-Chol show stable
monomodal distributions, with a diameter in agreement with that
imposed by the extrusion (100 nm) and low values of PDI, except for the
sample containing GL4 and RSV (PDI 0.3). Contrary to what observed in
DPPC/Chol liposomes, in this case the NNLS analysis confirms the
presence of a single population of liposomes, despite GL4 is included at a
L. Pagano et al.
Fig. 2. Growth curves of slime positive S. epidermidis treated with empty or resveratrol loaded liposomes. a) DPPC liposomes; b) DPPC/Chol liposomes; c) DPPC/
Chol/GL4 liposomes; d) DPPC/Chol/DC-Chol/GL4 liposomes. Each O.D. value was obtained subtracting O.D. of liposomes. RSV is 0.7 mM in all samples. Error bars
represent standard deviation of two independent experiments that were performed in triplicate (three independent wells), giving a total of six values.
concentration close to its cmc. This demonstrates the role of DC-Chol in
stabilizing the lipid bilayer contrasting the detergent activity of GL4.
DPPC and DPPC/Chol liposomes feature, as expected, a negative
value of ζ potential, due to the preferential exposure of the phosphate
groups of the phosphocholine. In the presence of RSV, the ζ-potential of
DPPC liposomes increases; this effect can be explained on the basis of the
localization of RSV in tight packed membranes, such as those composed
of DPPC. In fact, RSV displays a small ability in penetrating DPPC
membranes, and it is located mainly in the headgroup region, near to the
glycerol backbones of phospholipids, forming hydrogen bonds with
ester groups of the phosphate (53%) and of carbonyl moieties (47%) (Fei
et al., 2018). The geometry of the system imposes that with the aromatic
ring of RSV placed near the phosphate, the cationic choline moiety is
forced to move towards the surface, with an overall change of exposed
charges. The increment of the ζ-potential upon RSV addition is not
observed in DPPC/Chol liposomes. Probably in this case both play a role.
The ζ-potential values of glucosylated liposomes are positive and, on
the contrary of what observed for DPPC, the presence of RSV induces a
slight decrease in the absolute value, as previously observed in DOPC/
Chol/GL4 liposomes (Aiello et al., 2021). In this case, RSV could be
located in the membrane in a more hydrated environment and, there­
fore, in a small percentage could be ionized, with the negatively charged
hydroxyl group oriented towards the lipid water interface, thus making
the potential less positive. The more hydrated environment of RSV could
be ascribable to two different reasons: a different position of RSV in the
bilayer, favored by the interaction with the positively charged tetraal­
kylammonium moieties of GL4 (previously reported for polyphenols
(Yanes et al., 2000)); a higher hydration of the bilayer as a whole due to
6
Chemistry and Physics of Lipids 243 (2022) 105174
the presence of the polioxoethylenic spacer and to the formation of
hydrogen bonds between the GL4 ammonium groups and water.
All liposomes containing DC-Chol display positive and quite high
values of ζ-potential, both in the presence and in the absence of RSV. As
previously observed for DPPC/Chol/GL4 liposomes, a decrease in the
absolute value of ζ-potential is observed in the presence of RSV, more
pronounced for the formulation with GL4. The diameter of these vesicles
is around 70 nm, lower than expected; this effect has been observed also
in the case of mixed liposomes formulated with GL4, Chol and DOPC)
(Aiello et al., 2021), and was also reported in the case of liposomes
composed of glycosylated lipids and natural lipids like EPC (Latxague
et al., 2011) or DPPC-PE mixtures (Miyazawa et al., 2018).
All formulations feature quite high entrapment efficiencies (Fig. 1,
panels D, H). Interestingly, the cationic formulations containing either
the glucosylated amphiphile and/or DC-Chol, show higher values of EE
% with respect to DPPC/Chol formulations at the same concentration.
The same behavior was observed previously in the case of DOPC based
formulations including a glycosylated lipid bearing an ammonium
function (Aiello et al., 2021): this might be due to a favorable association
between RSV and quaternary ammonium function of glycosylated lipids,
and this hypothesis is supported by the observation that polyphenols
may associate with positively charged tetraalkylammonium moieties
(Yanes et al., 2000).
The antimicrobial activity of RSV-loaded liposomes was compared
with that of free RSV on two cell lines of S. epidermidis, a slime positive
strain (i.e. a strain able to produce the EPS components and hence able
to form a biofilm) and a slime negative strain.
The antimicrobial activity was evaluated following the bacterial
L. Pagano et al.
Fig. 3. Growth curves of slime negative S. epidermidis threated with empty or resveratrol loaded liposomes. a) DPPC liposomes; b) DPPC/Chol liposomes; c) DPPC/
Chol/GL4 liposomes; d) DPPC/Chol/DC-Chol/GL4 liposomes. Each O.D. value was obtained subtracting O.D. of liposomes. RSV is 0.7 mM in all samples. Error bars
represent standard deviation of two independent experiments that were performed in triplicate (three independent wells), giving a total of six values.
growth curves in the presence of RSV, either free or delivered by lipo­
somes. Growth curves were obtained measuring every 2 h the optical
density of liquid cultures incubated at 37 ◦ C. This technique is based on
the correlation between turbidity of bacterial solution and the number of
bacterial cells in the solution (Maier Pepper et al., 2000).
It is known that the surface of S. epidermidis is characterized by a high
number of glucose receptors (Iancu et al., n.d) that could be specifically
targeted by the glucose moieties decorating the surface of liposomes
(Iancu et al., n.d; Otto, 2009). Therefore, among all characterized for­
mulations, we chose to use a formulation containing only GL4, a
formulation containing both GL4 and DC-Chol and a formulation con­
taining only DC-Chol, in order to investigate both the role of glucose
functionalization on liposome surface and the cationic surface charge.
DPPC liposomes and a DPPC/Chol liposomes were investigated as well,
as reference of neutral and not-functionalized liposomes (Table 1).
In the first experiments RSV concentration was set at 0.7 mM, i.e.at
the MIC50 (Minimum Inhibitory Concentration required to inhibit the
growth of 50% of organisms) of free RSV for S. epidermidis according to
Kolouchová et al. (2018). Blank samples (in the absence of RSV) were
diluted to have lipid concentration equal to that of RSV-loaded
liposomes.
The plots in Fig. 2 and 3 show the growth curves of the strains of
S. epidermidis in the presence of RSV-loaded liposomes and in the pres­
ence of empty liposomes at the same lipid concentration. The growth of
untreated S. epidermidis (control) and the growth of the microorganism
after administration of free RSV are reported for comparison in each
plot.
7
Chemistry and Physics of Lipids 243 (2022) 105174
We observed that some samples show a higher O.D. with respect to
the control. This could be explained by the concomitant occurrence of
two possible phenomena: i) bacteria use liposome components as nu­
triments, thus showing a more intense cell growth; this can happen
especially in “low nutrient conditions” as the ones used in the experi­
ments; ii) in the samples containing both cationic liposomes and nega­
tively charged dead bacterial cells, flocculation may occur, thus
increasing the OD (Campanhã et al., 1999). In the case of cationic
DPPC/Chol/DC-Chol liposomes, both RSV-loaded and empty, the
turbidity of the samples was high and made the interpretation of the
results very difficult (data reported in the SI).
In the experiments on slime positive strain we observed that neutral
DPPC/RSV liposomes are not able to significantly inhibit the growth of
S. epidermidis (Fig. 2a) with respect to empty liposomes, at least for the
first 24 h. On the other hand, neutral DPPC/Chol/RSV liposomes show
an inhibitory effect after 10 h (P < 0.0001) whereas empty DPPC/Chol
liposomes do not show any inhibitory effect (Fig. 2b). This result sug­
gests that the addition of cholesterol to the liposomes might favor the
delivery of loaded RSV to bacterial cells. The higher value of OD
observed in the case of empty DPPC/Chol liposomes can be explained by
considering that both lipid component can serve as nutrients for bacte­
ria, as mentioned above.
Cationic glycosylated DPPC/Chol/GL4 liposomes gave interesting
results (Fig. 2c), as empty liposomes show low inhibition only after 22 h,
whereas RSV loaded liposomes show bacterial growth inhibition
immediately after the addition of liposomes (P < 0.0001), similarly to
that observed in the case of administration of free RSV. This is a key
L. Pagano et al.
Fig. 4. Growth curves of slime positive S. epidermidis treated with empty and resveratrol loaded DPPC/Chol/GL4 liposomes at decreasing resveratrol concentrations:
a) resveratrol 0.7 mM; b) resveratrol 0.35 mM; c) resveratrol 0.14 mM; d) resveratrol 0.07 mM. Each O.D. value was obtained subtracting O.D. of liposomes. Error
bars represent standard deviation of two independent experiments that were performed in triplicate (three independent wells), giving a total of six values.
result as this formulation is able to deliver RSV to bacterial cells without
being toxic by itself.
On the other hand, the formulation containing both GL4 and DC-
Chol resulted toxic in the absence of RSV (Fig. 2d),probably due to the
high cationic charge on liposomes’ surface combined with the detergent
properties of GL4.
The experiments on the slime negative strain showed results similar
to those obtained on the slime positive strain, the most significant dif­
ference concerning the experiments with DPPC/Chol/GL4 liposomes. In
fact, the slime negative strain resulted to be more sensitive to the
presence of the empty glycosylated liposomes compared to the slime
positive strains as indicated by the slight inhibition at the beginning of
the growth (up to 6 h, P < 0.0001), while RSV-loaded DPPC/Chol/GL4
liposomes were able to completely inhibit bacterial growth (P < 0.0001)
(Fig. 3c).
The high inhibitory effect of some of the formulations was observed
at 0.7 mM RSV,therefore we investigated the effect of the most prom­
ising formulations at lower concentrations of RSV, because the inclusion
of drugs inside liposomes should enhance their effect, thus decreasing
the value of concentration showing a therapeutical effect.
The growth curve of S. epidermidis was thus measured in the presence
of diluted formulations of DPPC/Chol, DPPC/Chol/GL4 and DPPC/
Chol/DC-Chol/GL4at 0.35 mM, 0.14 mM and 0.07 mM RSV (respec­
tively a half, a fourth, a tenth MIC50).
RSV-loaded DPPC/Chol liposomes feature a slight inhibitory effect
only at half MIC50, being ineffective at lower concentrations on both
8
Chemistry and Physics of Lipids 243 (2022) 105174
slime positive and slime negative strains (See SI); on the contrary, the
results obtained with DPPC/Chol/GL4 liposomes gave very interesting
results (Figs. 4 and 5).
In the experiments on slime positive strain, empty liposomes do not
show any inhibitory effect on the growth at lower concentrations (0.14
and 0.07 mM), whereas RSV-loaded liposomes feature an inhibitory
effect similar to free resveratrol at 0.35 mM (P = 0.038) (Fig. 4b).
Moreover, at lower concentration RSV-loaded liposomes showed a slight
inhibitory effect after some time from the mixing while free resveratrol
showed no effect at all (P < 0.0001, for time points > 10 h) (Fig. 4c and
d).
Results of the experiments on slime negative strain are somewhat
different. In the 0.7 mM experiments we observed an inhibitory effect
for both the empty and the loaded liposomes. This effect was still present
even at lower concentrations (P < 0.0001) (Fig. 5b–d) suggesting that
the slime negative bacterial cells, that are not able to produce EPS ma­
terials, are more susceptible to the cationic charge of the liposomes and
to the detergent effect of GL4.
The formulations comprising both GL4 and DC-Chol, empty and
RSV-loaded, display a very high toxicity, on both slime positive and
slime negative strains, in all the concentration range explored (data not
shown); as previously explained, this high toxicity is probably due to the
high cationic charge on liposomes’ surface combined with the detergent
properties of GL4.
L. Pagano et al.
Fig. 5. Growth curves of slime negative S. epidermidis treated with empty and resveratrol loaded DPPC/Chol/GL4 liposomes at decreasing resveratrol concentrations:
a) resveratrol 0.7 mM; b) resveratrol 0.35 mM; c) resveratrol 0.14 mM; d) resveratrol 0.07 mM. Each O.D. value was obtained subtracting O.D. of liposomes. Error
bars represent standard deviation of two independent experiments that were performed in triplicate (three independent wells), giving a total of six values.
4. Conclusions
In this work, we loaded RSV in cationic liposomes, in the presence
and in the absence of the glucosylated amphiphile GL4, following two
different liposome preparation protocols, the sonication and extrusion
ones. The results of HPLC (EE%) and DLS measurements (size and size
distribution) indicate that the extrusion procedure is certainly to be
preferred when GL4 is enclosed in the formulation; in fact, in this case
only the extrusion protocol allows obtaining monodisperse liposome
suspensions.
We then evaluated the antimicrobial activity of RSV-loaded lipo­
somes against two cell lines of S. epidermidis, a slime positive strain (i.e. a
strain able to produce the EPS components and hence able to form a
biofilm) and a slime negative strain.
The most interesting results of microbiological tests are those on the
slime positive strain in the presence of the glucosylated formulation
DPPC/Chol/GL4 under the MIC; in fact, these liposomes, without being
toxic by themselves, greatly improve the antibacterial efficacy of RSV
that is able to kill bacteria even at concentration tenfold under the MIC.
Therefore, the use of the cationic glucosylated amphiphile GL4, that
features both a cationic charge and a ligand recognized by glucose re­
ceptors of S. epidermidis, has allowed to achieve a non-toxic delivery
system able to target bacteria, to efficiently entrap RSV and to improve
its biological activity.
9
Chemistry and Physics of Lipids 243 (2022) 105174
Declaration of competing interest
The authors report no declarations of interest.
Acknowledgments
This work was in part supported by PRIN project (call 2018)
“BacHounds: Supramolecular nanostructures for bacteria detection”
(Prot. 2017E44A9P_004) funded by the italian Ministry of University
and Research (MUR).
L.P. and B.S. are grateful to Sapienza University of Rome, Call for
“Borse Di Studio Per Tesi Di Laurea All’estero-2017” for financial
support.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.chemphyslip.2022.105174.
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