Enhanced cellular uptake by "pharmaceutically oriented devices" of new simplified analogs of Linezolid with antimicrobial activity

Article abstract of DOI:10.1016/j.ijpharm.2013.11.048

The aim of the present study was to enhance cellular uptake of simplified analogs of Linezolid by their incorporation into suitable delivery devices in order to improve the antimicrobial activity of these novel synthesized oxazolidin-2-one derivatives. Th

Full text of DOI:10.1016/j.ijpharm.2013.11.048

International Journal of Pharmaceutics 461 (2014) 163–170
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Enhanced cellular uptake by “pharmaceutically oriented devices” of
new simplified analogs of Linezolid with antimicrobial activity
Ortensia Ilaria Parisia,d,1, Marco Fiorilloa,1, Anna Carusoa,d,1, Anna Rita Cappelloa,
Carmela Saturninob, Francesco Puocia,∗, Antonella Pannoa, Vincenza Dolcea,
Hussein El-Kashefc, Maria Stefania Sinicropia
a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende (CS), Italy
b Department of Pharmaceutical and Biomedical Sciences, University of Salerno, 84084 Fisciano (SA), Italy
c Department of Chemistry, Faculty of Science, Assiut University, 71516, Assiut, Egypt
d Department of Computer Engineering, Modeling, Electronics and Systems, University of Calabria, 87036 Rende (CS), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of the present study was to enhance cellular uptake of simplified analogs of Linezolid by their
incorporation into suitable delivery devices in order to improve the antimicrobial activity of these novel
synthesized oxazolidin-2-one derivatives.
The oxazolidin-2-one derivatives were synthesized by developing a rather simple one-pot reaction
starting from oxiranylmethanol and several primary amines. Three delivery devices were prepared by
following different synthetic approaches, such as single-step free radical grafting, precipitation poly-
merization and nano-emulsion. Finally, the antimicrobial activity of the novel synthesized compounds,
without any vehicle and after their incorporation into the delivery devices, was evaluated against
Escherichia coli and Saccharomyces cerevisiae by performing time-kill analyses.
The synthesized oxazolidinones exhibited modest antimicrobial activity against E. coli and S. cere-
visiae (MIC 16 ␮g/mL). A good activity was, instead, highlighted after their incorporation into the
prepared delivery devices (lecithin-based nano-emulsion, poly(N-vinyl-pyrrolidone)-methacrylic acid
grafted copolymer and spherical polymeric nanoparticles) (MIC ≤ 4 ␮g/mL). The incorporation into suit-
able vehicles, indeed, reduced by 4 times the normal MICs of the newly synthesized oxazolilidin-2-ones
and represents an effective strategy to overcome cellular penetration constraints.
Received 4 October 2013
Received in revised form
19 November 2013
Accepted 23 November 2013
Available online 1 December 2013
Keywords:
Drug carriers
Enhanced cellular uptake
Oxazolidinones
Antimicrobial activity
Simplified analogs of Linezolid
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
oxazolidinones (e.g. Linezolid, Fig. 1.) with the 50S A-site pocket at
the peptidyltransferase center (PTC) of the ribosome, which over-
The oxazolidinones, a new class of synthetic clinically important
antimicrobial agents, are active against the sensitive and resistant
Gram +ve organisms.
laps the aminoacyl moiety of an A-site bound tRNA, inhibiting
the protein synthesis process in whole cells of Gram +ve bacteria
(Wilson et al., 2008). On the other hand, the inhibition of protein
synthesis of Gram −ve bacteria, such as Escherichia coli (E. coli), has
been observed only at higher doses (Muller and Schimz, 1999).
Keeping in mind the aforementioned interesting antimicro-
bial activities of oxazolidinones and that the infections caused by
Gram −ve bacteria are emerging nowadays (Michalska et al., 2013),
the preparation of some new oxazolidin-2-ones potentially active
against this kind of microorganisms and investigate their formula-
tion and cellular uptake could be of relevant interest.
Initial studies designed to elucidate the oxazolidinones mech-
anism of action evidenced that these drugs inhibit the formation
of the initiation complex in bacterial translation systems by pre-
venting the formation of the N-formylmethionyl-tRNA ribosome-
mRNA ternary complex (Swaney et al., 1998). The identification of
the drug-binding site at the ribosome, as well as the mechanism
of action of these antimicrobial agents, is not fully under-
stood. However, recent works have highlighted the interaction of
Since the main target sites for antibacterial agents are located
within the cell, at the cytoplasmic membrane level or within the
cytoplasm, the wall structure of Gram −ve bacteria offers a complex
barrier system to these compounds (Denyer and Maillard, 2002).
The phenomenon of cellular impermeability, indeed, is due to the
presence of an outer envelope represented by a bilayer structure
∗
Corresponding author. Tel.: +39 0984 493151; fax: +39 0984 493298.
E-mail addresses: francescopuoci@gmail.com, francesco.puoci@unical.it
(F. Puoci).
1
These authors equally contributed to the manuscript.
0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijpharm.2013.11.048

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O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
Corporation, New York USA) at 25.0 0.1 ◦C by measuring the auto-
correlation function at 90◦. The laser was operating at 658 nm.
2.2. Synthesis of simplified analogs of Linezolid
2.2.1. Preparation of (RS)-3-alkyl (or
Fig. 1. Chemical structure of Linezolid.
aryl)-5-hydroxymethyloxazolidin-2-ones (5a,b) (general method)
To a stirred solution of the appropriate primary amines 3a,b
(8.62 mmol) in MeOH (3 mL), (RS)-oxiranylmethanol 2 (7.84 mmol)
was slowly added. The reaction mixture was stirred further at room
temperature for 12 h to give intermediates 4a,b. Diethylcarbonate
(9.09 mmol) and anhydrous MeONa (0.74 mmol) were then added
and the resulting mixture was heated under reflux for 12 h. After
cooling to room temperature, the reaction mixture was evaporated
under reduced pressure to afford an oil residue. This oil was taken
up with EtOAc and the organic layer washed with water. Then it
was separated, dried (Na2SO4) and concentrated under reduced
pressure to give a solid or oil product, which was purified using
silica gel column chromatography.
consisting of lipopolysaccharides and phospholipids. Thus, antimi-
crobial agents have to cross this outer membrane in order to get
their principal target site.
Limited cellular penetration could reduce the effectiveness of
many antimicrobial treatments and, in view of this fact, the present
study was focused on the exploration of antimicrobial activity of the
synthesized therapeutic agents alone and after their incorporation
into “pharmaceutically oriented devices”.
In order to modulate the cellular uptake of these compounds,
three different strategies were adopted. The novel synthesized
oxazolidin-2-ones were, indeed, incorporated into drug delivery
carriers of different nature and structure, such as lecithin-
based nano-emulsion, poly(N-vinyl-pyrrolidone)-methacrylic acid
grafted copolymer (PVP-MAA) and spherical nanoparticles (SNs).
The antimicrobial activity of the compounds, without any vehi-
cle and after their incorporation into the delivery devices, was
evaluated against E. coli and Saccharomyces cerevisiae (S. cerevisiae)
by performing time-kill analyses according to the method of the
Clinical and Laboratory Standards Institute (Clinical and Laboratory
Standards Institute, 1999).
2.2.2. (RS)-3-Allyl-5-hydroxymethyl-oxazolidin-2-one (5a)
White solid, using CHCl3:MeOH (9:1) as eluent, yield 45%; mp
82 ◦C. IR spectrum, ꢀ, cm−1: 3413, 2924, 1733, 1645, 1263, 765.
1H NMR spectrum (CDCl3), ı, ppm: 3.18 (1H, br.s, OH); 3.33–3.61
(4H, m, CH2NCH2); 3.77–3.86 (2H, m, CH2OH); 4.51–4.59 (1H, m,
CHCH2OH); 5.14–5.26 (2H, m, CH2CH CH2); 5.63–5.78 (1H, m,
CH2CH CH2). 13C NMR spectrum (CDCl3), ı, ppm: 154.69; 133.85;
119.54; 76.03; 67.38; 59.61; 42.60. Mass spectrum (EI, 70 eV), m/z
(Irel, %): 157 [M]+ (100), 126 (74), 98 (89), 82 (98), 68 (88), 56 (44).
Found, %: C 53.46; H 7.01; N 8.88. C7H11NO3. Calculated, %: C 53.49;
H 7.05; N 8.91.
The choice of these kinds of vectors, characterized by such
a different construction, allows to hypothesize several possible
mechanisms involved in the enhancement of cellular uptake and,
consequently, in the improvement of the antimicrobial activity.
2.2.3. (RS)-5-Hydroxymethyl-3-phenethyl-oxazolidin-2-one (5b)
White solid, using EtOAc:hexane (4:1) as eluent, yield 50%;
mp 93 ◦C. IR spectrum, ꢀ, cm−1: 3444, 2927, 1735, 1603, 752. 1H
NMR spectrum (CDCl3), ı, ppm (J, Hz): 2.78–2.86 (2H, t, CH2Ph);
3.22–3.53 (6H, m, CH2NCH2, CHOH, OH); 3.69–3.77 (1H, dd, J = 3.30;
J = 12.60, CHOH); 4.41–4.50 (1H, m, CHCH2OH); 7.11–7.26 (5H, m,
Ar). 13C NMR spectrum (CDCl3), ı, ppm: 158.16, 138.25, 128.66,
128.59, 126.58, 73.72, 62.64, 46.09, 45.35, 33.72. Mass spectrum
(EI, 70 eV), m/z (Irel, %): 221 [M] + (36), 130 (100), 104 (55), 77 (19),
56 (18). Found, %: C 65.18; H 6.79; N 6.29. C12H15NO3. Calculated,
%: C 65.14; H 6.83; N 6.33.
2. Materials and methods
2.1. Materials and instrumentation
Commercial reagents were purchased from Aldrich, Acros
Organics and Alfa Aesar and were used without additional purifi-
cation.
Melting points were determined on a Gallenkamp melting point
apparatus.
The IR spectra were recorded on a Fourier Transform Infrared
Spectrometer FT/IR-4200 for KBr pellets.
GC/MS analyses were performed using a 17AA-V3 230 VLV spec-
trometer or a 6890N Network GC System (Agilent Technologies Inc.,
Palo Alto, CA, USA) equipped with an HP-5MS (30 m × 0.25 mm,
PhMesiloxane 5%) capillary column. The mass detector was oper-
ated in the ionization chemical mode (CI-CH4).
2.3. Synthesis of “pharmaceutically oriented delivery devices”
2.3.1. Poly(N-vinyl-pyrrolidone)-methacrylic acid (PVP-MAA)
grafted copolymer
Single-step grafting of methacrylic acid (MAA) onto poly(N-
vinyl-pyrrolidone) (PVP, average M.W. 360,000, Sigma–Aldrich)
was carried out by employing hydrogen peroxide/ascorbic acid as
biocompatible and water soluble redox pair (Parisi et al., 2013).
In a 25 mL glass tube, 4.0 g of PVP were dissolved in 15 mL of dis-
tilled water, then 1 mL of distilled MAA and 0.5 mL of H2O2 1.0 M
containing 0.025 g of ascorbic acid were added. The mixture was
maintained under stirring at 25 ◦C for 3 h under atmospheric air.
The resulting PVP-MAA grafted copolymer was purified by succes-
sive washing steps with distilled water, recovered by filtration and,
finally, dried overnight in a vacuum oven set at 40 ◦C.
An amount of the synthesized PVP-MAA copolymer (128 mg)
was immersed in 7.7 mL of a phosphate buffer solution (pH 7.4).
Then, 0.3 mL of a solution in DMSO containing 32 mg of the antimi-
crobial agent were added and the obtained mixture was sonicated
for 30 min.
ESI-MS was performed using a spectrometer LC–MS Waters
alliance 2695 (ESI+).
1H NMR (300 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Bruker 300 spectrometer. Chemical shifts are
expressed in parts per million downfield from tetramethylsilane
as an internal standard.
Thin layer chromatography (TLC) was performed on silica gel
60F-264 (Merck).
The scanning electron microscopy (SEM) photographs were
obtained with a Jeol JSMT 300 A; the surface of the samples was
made conductive by the deposition of a gold layer on the samples
in a vacuum chamber. Approximate range in particle size was deter-
mined employing an image processing and analysis system, a Leica
DMRB equipped with a LEICA Wild 3D stereomicroscope.
Dynamic light scattering (DLS) analysis was performed using
a
90 Plus Particle Size Analyzer (Brookhaven Instruments

O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
165
Scheme 1. Synthesis of oxazolidinones 5a and 5b.
2.3.2. Spherical nanoparticles (SNs)
Test organisms incubated in appropriate medium over night at
30 ◦C (yeast) or 35 ◦C (bacteria) were diluted with fresh medium to
ca. 104 CFU/mL and the diluted cultures were pre-incubated for 2 h.
Each drug, solubilized in dimethyl sulfoxide (DMSO) was added to
the cultures at concentrations of 1× MIC. Samples (0.1 mL) of the
cultures were removed at 0, 2, 4, 6 and 24 h of incubation and serial
10-fold dilutions were prepared in saline. The number of viable cells
was determined as described in the literature (Jung et al., 2012).
Spherical polymeric nanoparticles were prepared by precipi-
tation polymerization employing methacrylic acid and ethylene
glycol dimethacrylate (EGDMA) as functional monomer and cross-
linking agent, respectively (Puoci et al., 2004).
In a 100 mL round bottom flask, distilled MAA (8 mmol) were
dissolved in a mixture of acetonitrile (20 mL) and methanol (20 mL),
then EGDMA (10 mmol) and 2,2-azoisobutyronitrile (AIBN, 50 mg)
were added. The polymerization mixture was degassed in a son-
icating water bath, purged with nitrogen for 10 min cooling with
an ice bath. The flask was then gently agitated (40 rpm) in an oil
bath. The temperature was increased from room temperature to
60 ◦C within 2 h, and then kept at 60 ◦C for 24 h. At the end of the
reaction, the resulting nanoparticles were filtered, washed with
100 mL of ethanol, 100 mL of acetone and 100 mL of diethyl ether
and, successively, dried overnight under vacuum at 40 ◦C.
An amount of the resulting SNs (128 mg) was immersed in 3 mL
of a solution in ethanol containing 32 mg of the antimicrobial agent
and soaked for 3 days at room temperature. Then the solvent was
removed under reduced pressure.
3. Results and discussion
3.1. Chemistry
Different methods for the synthesis of oxazolidin-2-one com-
pounds were reported in the literature (Lamanna et al., 2004; Osa
et al., 2005; Robles-Machín et al., 2006; Ella-Menyea and Wang,
2007; Adibpour et al., 2010; Lancelot et al., 1993, 1996; Saturnino
et al., 2000, 2004).
In the present study, we report
a rather simple one-
pot synthesis of two compounds (5a,b) starting from racemic
(RS)-oxiranylmethanol (2) and different primary amines (3a,b)
(Scheme 1). Thus, compound 2 was allowed to react with differ-
ent primary amines (3a,b) in methanol, to give the corresponding
3-substituted-amino-1,2-propanediols intermediates (4a,b). The
latter were then treated with equimolar amounts of diethylcar-
bonate in the presence of catalytic amounts of sodium methoxide
to give N-substituted-5-hydroxymethyl-oxazolidin-2-ones (5a,b)
in 45–87% yield (Table 1).
2.3.3. Nano-emulsion based on soybean lecithin
Nano-emulsion containing the antimicrobial agent was pre-
pared by the following procedure.
The emulsifier and the emulsifier adjuvant, such as soybean
lecithin (1.2%, w/w) and tween 80 (1.2%, w/w) respectively, were
added to soybean oil as the oil phase (10%, w/w). Lecithin was
stirred with heating to 80 ◦C until it was completely dissolved
in the oil phase and the obtained mixture was cooled to room
temperature. Then, 0.3 mL of a solution in DMSO containing the
antimicrobial agent (0.24%, w/w) was added and mixed homoge-
neously. The mixture was added dropwise into a 2.25% glycerol
solution preheated at 40 ◦C, and then homogenized for 10 min at
24,000 rpm using a homogenizer. After the completion of the emul-
sification, the pH was adjusted to neutrality with NaOH (Kim and
Lee, 1999).
3.2. Synthesis of “pharmaceutically oriented delivery devices”
3.2.1. PVP-MAA grafted copolymer
Methacrylic acid was grafted onto a preformed polymeric back-
bone, such as poly(N-vinyl-pyrrolidone), by a free radical procedure
involving a one-pot reaction at room temperature. Hydrogen per-
oxide/ascorbic acid redox pair was employed as water-soluble and
biocompatible initiator system allowing to perform the grafting
process in aqueous media. In view of this fact, the use of organic sol-
vents and the generation of any kind of toxic reaction byproducts
are avoided.
2.4. Biological analyses
2.4.1. Yeast and bacterial strains
Yeast strains Y10000, wild-type, was provided by the EUROFAN
resource center EUROSCARF (Frankfurt, Germany). The cells were
grown in rich medium containing 2% Bactopeptone and 1% yeast
extract (YP), supplemented with fermentable 2% glucose carbon
sources (Iacopetta et al., 2010). The final pH was adjusted to 4.8.
The E. coli strains ATCC 8739 was provided by REMEL. The cells
were grown in LB medium containing 10 g/L Tryptone, 5 g/L yeast
extract and 0.5 g/L NaCl (Iacopetta et al., 2010; Santoro et al., 2011).
The final pH was adjusted to 7.4.
Minimum inhibitory concentrations (MICs) were determined by
the agar dilution method as described by the Clinical and Labora-
tory Standards Institute (CLSI) (Clinical and Laboratory Standards
Institute, 2012).
The mechanism of redox pair reaction (Fig. 2.) consists of the
oxidation of ascorbic acid (AA) by hydrogen peroxide with the for-
mation of hydroxyl and ascorbate radicals (Curcio et al., 2009).
Hydroxyl radical represents the most reactive radical among the
reactive oxygen species and its reaction with PVP chain gener-
ates macroradicals centered in three possible positions according
to the lability of the hydrogen atoms present in the polymeric
Table 1
Synthesized oxazolidinones.
Cpd
5a
RNH2
Rdt %
45
NH2
3a
2.4.2. Time-kill studies
Time-kill analyses were performed according to the method of
the CLSI (Clinical and Laboratory Standards Institute, 1999).
NH2
5b
3b
50

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O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
Precipitation polymerization represents a simple and efficient
method to obtain uniform polymeric particles in the absence of any
stabilizer. The size and size distribution of particles are involved in
size-dependent cellular uptake (Zhang et al., 2009) and this poly-
merization technique allows to have a better control on size and
shape.
The polymerization system consists of only monomer, cross-
linker, initiator and solvent as components (Li and Stover, 1993)
and, in the first stage of the process, monomers form oligomer
radicals. Then, the formed oligomers crosslink and the obtained
crosslinked nuclei aggregate into larger particles leading to the
formation of the final polymer beads.
In a precipitation polymerization process, the adopted concen-
trations of monomer, cross-linking agent and initiator affect the
size of synthesized particles. The diameter of polymeric spheres,
indeed, increases with increasing monomer or initiator concen-
tration (Puoci et al., 2004); on the other hand, the particles size
decreases as the cross-linker percentage increases (Shim et al.,
2004).
In the present study, spherical nanoparticles (SNs) were pre-
pared by precipitation polymerization employing MAA, EGDMA
and AIBN as functional monomer, cross-linking agent and initia-
tor, respectively. For this purpose, in the prepolymerization feed
the employed MAA/EGDMA molar ratio was equal to 8:10 (Cirillo
et al., 2009). This ratio represents, indeed, the optimal value to
obtain nanospheres with the desired characteristics such as spher-
ical shape and nanometer size.
Fig. 2. Mechanism of AA/H2O2 redox pair reaction.
structure. Literature (Barros et al., 2006), indeed, reported that C
H
bonds of PVP ␣-positioned to a heteroatom or a carbonyl are lower
in energy, mainly due to the stabilization of the radical product.
Generated macroradicals react with MAA monomer which is graft-
copolymerized onto the PVP preformed polymeric chain.
Poly(N-vinyl-pyrrolidone) is a polymeric material characterized
by chemical stability, low toxicity, biocompatibility with living tis-
sues, general bioinertness and water solubility due to the presence
of the polar lactam group which increases its hydrophilicity (Liu
et al., 2013). On the other hand, the non-polar methylene groups
make this material lipophilic. All these features make PVP widely
employed as biomaterial and excipient in various fields, such as
pharmaceutical, cosmetic, biomedical and food.
The grafting of hydrophilic molecules onto the surface of a
material could improve its biocompatibility regulating protein
adsorption and cell adhesion. In this study, the conjugation of PVP
with methacrylic acid aimed to increase the hydrophilicity of the
copolymer, providing pH-sensitivity to the system, and the choice
of MAA was dictated to the broad applications of methacrylate
polymers in biotechnology and biomedicine (Pérez et al., 2006).
PVP-MAA and the respective control polymer (PVP) were
characterized by Fourier Transform IR spectrophotometry. By com-
paring the two IR spectra (Fig. 3), the PVP-MAA spectrum (trace
B) showed the appearance of a new signal at 1717 cm−1 ascrib-
able to the C O stretching vibration of the carboxylic groups of
methacrylic acid.
Spherical geometry and the practical monodispersion of the
prepared nanospheres were confirmed by scanning electron micro-
graphs (Fig. 4).
3.2.3. Nano-emulsion based on soybean lecithin
An emulsion is a mixture of two immiscible liquids, such as
water and oil, with one dispersed in the other (Leal-Calderon et al.,
2007). The preparation of emulsions requires the use of surfac-
tants in the aim to decrease the interfacial tension and maintain
the stability of the formulation.
3.2.2. Spherical nanoparticles (SNs)
Nano-emulsions represent promising drug delivery vehicles due
to their small size, biocompatibility, relative stability and ability to
protect drugs from hydrolysis and enzymatic degradation under
physiological conditions (Jafari et al., 2007).
In the last years, polymeric nanoparticles have received consid-
erable attention due to their potential applications as drug delivery
systems.
Fig. 3. FT-IR spectra of PVP (a) and PVP-MAA (b).

O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
167
Fig. 4. Scanning electron micrograph of SNs.
In this report, nano-emulsions containing the antimicrobial
agents were prepared using soybean lecithin and tween 80 as
emulsifier and emulsifier adjuvant, respectively. The droplet size
distribution was measured by dynamic light scattering (DLS) and it
was found to be 259.6 nm (effective diameter) with a PDI of 0.268
(Fig. 5).
3.3. Time-kill curves
The oxazolidinones represent a novel chemical class of synthetic
antimicrobial agents active against a large spectrum of sensitive
and resistant Gram +ve organisms due to a unique mechanism of
protein synthesis inhibition involving the binding at the P site of the
ribosomal 50S subunit (Bozdogan and Appelbaum, 2004). However,
protein synthesis of Gram −ve bacteria was affected only at higher
doses by these molecules.
In this study, new oxazolidin-2-ones potentially active against
Gram −ve bacteria were synthesized by following a simple one-step
reaction and their formulation and cellular uptake were investi-
gated.
Fig. 6. Time-kill curve profiles of 5a and 5b against E. coli (panel A) and S. cerevisiae
(panel B).
Two different kinds of microorganisms, such as E. coli and S.
cerevisiae, were chosen to evaluate the antimicrobial activity by
performing time-kill analyses. The first one was chosen because
it is a common type of bacteria and represents one of the most
Fig. 5. Droplet size distributions of lecithin-based nano-emulsion determined by dynamic light scattering.

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O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
Fig. 8. Time-kill curve profiles of 5a and 5b against S. cerevisiae, after incorporation
into lecithin-based nano-emulsion (panel A), PVP-MAA (panel B) and SNs (panel C).
Fig. 7. Time-kill curve profiles of 5a and 5b against E. coli, after incorporation into
lecithin-based nano-emulsion (panel A), PVP-MAA (panel B) and SNs (panel C).
PVP-MAA copolymer is characterized by
a poly(N-vinyl-
pyrrolidone) backbone with polymethacrylic acid grafted side
chains attached to it. PVP is an interesting material from a bio-
logical point of view due to the presence of the lactam group in
the pyrrolidone moiety which makes its structural feature sim-
ilar to those of proteins (Liu et al., 2013). The performance of
the prepared grafted copolymer as drug device depends on its
lipophilicity/hydrophilicity ratio. Since membrane proteins also
have hydrophilic and hydrophobic domains, these structural fea-
tures may influence protein interactions with the copolymer.
Nano-emulsions containing the antimicrobial agents were,
instead, prepared using soybean lecithin and tween 80 as emul-
sifier and emulsifier adjuvant, respectively. The results obtained
employing this formulation could be attributed to its cellular per-
meability due to the presence of lecithin which is a complex
mixture containing phospholipids, the main components of the bio-
logical membranes. Furthermore, the use of delivery systems at the
nanoscale may potentially increases the passive cellular absorption
mechanisms allowing the antimicrobial agent to act from the inner
side on the cytoplasmic membrane.
frequent causes of many widespread bacterial infections; S. cere-
visiae was selected due to the construction of its cell wall which is
typical of other relevant and dangerous Saccharomycetes such as
Saccharomyces albicans.
The synthesized oxazolidin-2-ones (5a and 5b), without any
vehicle, showed modest antimicrobial activity against E. coli (Fig. 6,
panel A) and S. cerevisiae (Fig. 6, panel B) (MIC 16 ␮g/mL) in com-
parison with the results obtained using the microorganisms alone.
The problem could be probably due to the cellular imperme-
ability of these microorganisms to the antimicrobial agents. Poor
cellular uptake, indeed, could be responsible for the disappointing
activity against bacteria that the synthesized compounds exhibited.
In order to overcome this drawback, 5a and 5b molecules were
incorporated into three different carriers prepared according to
reported literature: nano-emulsion based on soybean lecithin (Kim
and Lee, 1999), PVP-MAA grafted copolymer (Parisi et al., 2013) and
spherical polymeric nanoparticles (Puoci et al., 2004).
We supposed that the chosen vehicles have the ability to
improve the cellular uptake of the synthesized oxazolidin-2-ones
derivatives by several possible mechanisms.
Finally, spherical nanoparticles (SNs) were prepared by
precipitation polymerization employing MAA and EGDMA as

O.I. Parisi et al. / International Journal of Pharmaceutics 461 (2014) 163–170
169
functional monomer and cross-linking agent, respectively. Poly-
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The synthesized derivatives showed modest antimicrobial
activity against E. coli and S. cerevisiae (MIC 16 ␮g/mL). As
hypothesized,
a good activity was instead highlighted after
their incorporation into the prepared delivery devices such as
lecithin-based nano-emulsion, PVP-MAA grafted copolymer and
SNs (MIC ≤ 4 ␮g/mL).
The obtained results clearly indicated that the incorporation
into suitable drug carriers could be an effective and very promising
strategy to enhance the cellular uptake of the synthesized thera-
peutic agents and, consequently, improve their antimicrobial effect.
The presence of vehicles, indeed, reduced by 4 times the normal
MIC values of the newly synthesized compounds improving their
antimicrobial activity against two different kinds of microorgan-
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Conflict of interest
Santoro, A., Cappello, A.R., Madeo, M., Martello, E., Iacopetta, D., Dolce, V.,
2011. Interaction of fosfomycin with the glycerol 3-phosphate transporter of
Escherichia coli. Biochim. Biophys. Acta 1810, 1323–1329.
The authors report no declarations of interest.
Saturnino, C., Buonerba, M., Capasso, A., Lancelot, J.C., De Martino, G., 2004. Eval-
uation of analgesic and anti-inflammatory profile of beta-lactam monocyclic
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Acknowledgments
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We wish to thank Italian Minister of University and Research for
financial support of this work.

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