Antibiotic-conjugated polyacrylate nanoparticles: New opportunities for development of anti-MRSA agents

Article abstract of DOI:10.1016/j.bmcl.2006.09.098

This report describes the preparation of polyacrylate nanoparticles in which an N-thiolated β-lactam antibiotic is covalently conjugated onto the polymer framework. These nanoparticles are formed in water by emulsion polymerization of an acrylated antibiotic pre-dissolved in a liquid acrylate monomer (or mixture of co-monomers) in the presence of sodium dodecyl sulfate as a surfactant and potassium persulfate as a radical initiator. Dynamic light scattering analysis and electron microscopy images of these emulsions show that the nanoparticles are approximately 40 nm in diameter. The emulsions have potent in vitro antibacterial properties against methicillin-resistant Staphylococcus aureus and have improved bioactivity relative to the non-polymerized form of the antibiotic. A unique feature of this methodology is the ability to incorporate water-insoluble drugs directly into the nanoparticle framework without the need for post-synthetic modification. Additionally, the antibiotic properties of the nanoparticles can be modulated by changing the length or location of the acrylate linker on the drug monomer.

Full text of DOI:10.1016/j.bmcl.2006.09.098

Bioorganic & Medicinal Chemistry Letters 17 (2007) 53–56
Antibiotic-conjugated polyacrylate nanoparticles:
New opportunities for development of anti-MRSA agents
Edward Turos,a,* Jeung-Yeop Shim,a Yang Wang,a Kerriann Greenhalgh,a
G. Suresh Kumar Reddy,a Sonja Dickeyb and Daniel V. Limb
aDepartment of Chemistry, 4202 East Fowler Avenue, CHE 207, University of South Florida, Tampa, FL 33620, USA
bDepartment of Biology, 4202 East Fowler Avenue, University of South Florida, Tampa, FL 33620, USA
Received 3 September 2006; revised 29 September 2006; accepted 29 September 2006
Available online 4 October 2006
Abstract—This report describes the preparation of polyacrylate nanoparticles in which an N-thiolated b-lactam antibiotic is cova-
lently conjugated onto the polymer framework. These nanoparticles are formed in water by emulsion polymerization of an acrylated
antibiotic pre-dissolved in a liquid acrylate monomer (or mixture of co-monomers) in the presence of sodium dodecyl sulfate as a
surfactant and potassium persulfate as a radical initiator. Dynamic light scattering analysis and electron microscopy images of these
emulsions show that the nanoparticles are approximately 40 nm in diameter. The emulsions have potent in vitro antibacterial prop-
erties against methicillin-resistant Staphylococcus aureus and have improved bioactivity relative to the non-polymerized form of the
antibiotic. A unique feature of this methodology is the ability to incorporate water-insoluble drugs directly into the nanoparticle
framework without the need for post-synthetic modification. Additionally, the antibiotic properties of the nanoparticles can be
modulated by changing the length or location of the acrylate linker on the drug monomer.
Ó 2006 Elsevier Ltd. All rights reserved.
The development of antibiotics for control of pathogen-
ic bacteria has been of pressing need in this era of drug-
resistant infections.1 N-Methylthiolated b-lactams have
recently been identified in our laboratory as a new fam-
ily of antibacterial agents active against Staphylococcus
bacteria, including methicillin-resistant Staphylococcus
aureus (MRSA).2 The compounds have also displayed
promising anticancer properties.3 Our recent studies
have suggested that these lactams exert their growth
inhibitory effects on bacteria through a mode of action
that is distinctively different to that of other b-lactam
antibiotics, and possess structure–activity patterns
unlike those already mapped for other b-lactam antibac-
terials such as the penicillins. One of the major limita-
tions in the potential application of these N-thiolated
b-lactam compounds, however, is their exceedingly low
water solubility.4 Thus, we were interested in identifying
an effective drug delivery platform that would enhance
the water solubility of the lactams, without sacrificing
inherent bioactivity.
Drug delivery vehicles such as liposomes and gold
nanoparticles have been developed to improve bioavail-
ability, efficacy, and specificity of pharmaceutical
compounds, particularly for anticancer agents, but
nanoparticles have received surprisingly little attention
in the antibiotic and infectious disease area.5,6 Some of
the few notable examples have included antibiotic-en-
capsulated polymeric nanoparticles and liposomes,7,8
biodegradable nanospheres,9,10 and surface-coated gold
and silver nanoparticles.11–13 This report describes the
development of antibacterial polyacrylate nanoparticles
based on well-precedented emulsion polymerization
procedures.
In this process, the antibacterial drug is first converted
to an acrylated derivative and then dissolved to homoge-
neity in a liquid acrylate monomer (or mixture of com-
patible liquid monomers) at 70 °C. This mixture is then
pre-emulsified in purified water containing 3% w/w sodi-
um dodecyl sulfate with rapid stirring (Scheme 1). The
resulting homogeneous solution of micelles is then treat-
ed with potassium persulfate (1% w/w), a water-soluble
radical initiator, to induce free radical polymerization.14
The resulting emulsion is found to contain uniformly
sized polyacrylate nanoparticles in which the drug
Keywords: b-Lactams; Nanoparticles; Nanobiotics; MRSA; Polyacry-
lates; Emulsion polymerization.
*
Corresponding author. Tel.: +1 813 974 7312; fax: +1 813 974
1733; e-mail: eturos@shell.cas.usf.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.098

54
E. Turos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 53–56
O
microemulsion
polymerization
O
O O
Drug
H3C
O
OC4H9
O
CH3
Drug
O
+
O-Drug
water, 70 o
C
O
O
O
Drugs 1-4
Nanoparticles NP1-4
~40 nm in diameter
Scheme 1.
O
O
SCH3
O
SCH3
OCH3
N
N
O
Cl
OCH3
H3CO
O
O
N
O
O
O
O
Cl
N
Cl
a
b
O
O
2
1
HO
O
O
8
O
SMe
Cl
O
O
SCH3
5
N
N
O
O
O
H
O
O
SMe
N
N
O
O
Cl
O
Cl
O
O
O
c
O
O
O
O
O
O
O
O
O
O
O
O
3
4
9
3
Figure 1. N-Thiolated b-lactam acrylate monomers 1–4.
Scheme 3. Reagents and conditions: (a) mono-2-acryloyloxyethyl
succinate, EDCI, DMAP, CH2Cl2, rt, 77%; (b) ceric ammonium
nitrate, H2O, CH3CN, 0 °C, 86%; (c) N-methylthiophthalimide, CH2
Cl2, Hunig’s base, reflux, 72%.
monomer is covalently incorporated directly into the
polymeric matrix of the nanoparticle. A unique feature
of this methodology is that the nanoparticle emulsion
containing the antibiotic agent is built from its monomer
constituents in one step, without the need for further
chemical modification or derivatization. Furthermore,
the nanoparticles display potent antibacterial activity
against MRSA.
bis-Acrylated compound 4 was synthesized starting with
p-hydroxybenzaldehyde and p-anisidine, as shown in
Scheme 4.
Initially, N-methylthio b-lactam monomer 1 was sub-
jected to emulsion polymerization conditions using ethyl
acrylate as a liquid acrylate monomer, which gave the
desired emulsion. However, upon prolonged storage,
For these studies, four N-thiolated b-lactam derivatives
were selected for use as antibiotic drug monomers in or-
der to assess the effect of different lengths and locations
of the acrylate linker on the resulting nanoparticle size
and anti-MRSA bioactivity (Fig. 1).
OCH3
OCH3
Lactam 1 was prepared as previously reported.3 Com-
pound 2 was synthesized in three steps from hydroxy
b-lactam 5 by O-acrylation and oxidative deprotection
followed by N-methylthiolation of the lactam (Scheme
2).
OCH3
OAc
CHO
OH
OCH3
NH2
O
a
b
N
c
N
N
+
AcO
12
OH
OAc
11
10
Compound 3 was prepared in three steps from 3-hy-
droxy b-lactam 5 by carbodiimide coupling of the alco-
hol moiety with mono-2-acryloyloxyethyl succinate,
then N-aryl deprotection and N-methylthiolation
(Scheme 3).
OCH3
OCH3
O
O
O
N
N
e
d
O
HO
O
O
OH
14
13
O
O
O
SMe
NH
N
O
O
f
g
OCH3
OCH3
O
4
O
O
O
O
H
O
O
O
O
O
SCH3
Cl
O
O
15
N
N
5
N
N
a
c
b
O
Cl
O
Cl
O
Cl
HO
Scheme 4. Reagents and conditions: (a) CSA, CH2Cl2, rt; (b) acetyl
chloride, Et3N, CH2Cl2, 92% overall; (c) acetoxyacetyl chloride, Et3N,
CH2Cl2, 65%; (d) KOH, MeOH, acetone, 0 °C, 92%; (e) acryloyl
chloride, Et3N, CH2Cl2, 93%; (f) ceric ammonium nitrate, CH3CN,
H2O, 0 °C, 84%; (g) N-methylthiophthalimide, Hunig’s base, CH2Cl2,
reflux, 72%.
2
6
7
Scheme 2. Reagents and conditions: (a) acryloyl chloride, NaH,
CH2Cl2, 85%; (b) ceric ammonium nitrate, CH3CN, H2O, 0 °C, 90%;
(c) N-methylthiophthalimide, CH2Cl2, Hunig’s base, reflux, 82%.

E. Turos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 53–56
55
Table 1. Agar dilution MIC values of nanoparticle emulsions NP0–4
(1% w/w drug in the emulsion) and b-lactam monomers 1–4 against
Staphylococcus aureus (ATCC 25923) and MRSA (ATCC 43300)
this emulsion was found to be sensitive to aggregation
and precipitation. Consequently, we examined other
combinations of acrylate monomers and found that a
mixture of butyl acrylate and styrene (in a 7:3 ratio by
weight) afforded the most stable emulsions. In each case,
these emulsions contained the polyacrylate (20% by
weight) comprised of the bulk monomers and one of
the acrylate lactams 1–4 (up to 5% by weight). Purifica-
tion of the emulsion was performed by continuous
extraction using ethyl ether followed by centrifugation
on a low-speed benchtop centrifuge. The purification en-
abled minor impurities such as excess surfactant or
residual monomers to be removed without altering the
physical characteristics or the antibacterial activity of
the nanoparticles in the emulsion. Analysis of the emul-
sions of nanoparticles NP0–4 by scanning electron
microscopy (SEM), transmission electron microscopy
(TEM), dynamic light scattering (DLS), and atomic
force microscopy (AFM) indicated the formation of
spherical nanoparticles having a uniform diameter of
30–50 nm (Fig. 2). The zeta potential of the emulsified
nanoparticle solutions registered between À50 and
À70 mV, reflecting a high anionic charge on the nano-
particle surface and high relative particle stability. Pro-
ton nuclear magnetic resonance (NMR) analysis of the
thin film produced by evaporation of the nanoparticle
emulsions confirmed that essentially all of the butyl
acrylate, styrene, and b-lactam components were incor-
porated into the skeletal framework of the polymer dur-
ing the polymerization.15
Sample
S. aureus
MRSA
Nanoparticles
NP0
NP1
>256
32
16
>256
32
16
NP2
NP3
8
16
8
16
NP4
Lactam monomers
1
2
3
4
128
128
64
128
128
64
128
128
MIC values refer to the lowest concentration of bound drug (lg/mL)
needed to completely inhibit bacterial growth for 24 h. MIC determi-
nations were run in triplicate and the values obtained for each com-
pound were invariant.
ticles, NP0, were completely devoid of antimicrobial
activity.
The MIC data in Table 1 also indicate that the antibiot-
ic-conjugated polyacrylate nanoparticles NP1–4 had en-
hanced (four to eightfold) anti-MRSA activity
compared to their acrylated drug monomers 1–4. This
heightened activity may be indicative of an increased
bioavailability (membrane permeability or enhanced lo-
cal concentration) of the antibiotic agent as a conse-
quence of the nanoparticle serving as a delivery
vehicle. Furthermore, antibacterial activity of the nano-
particles can be altered by changing the location or the
length of the acrylate linker. Thus, positioning the acry-
late functionality on the C3 center of the lactam mono-
mer, as shown for monomer 2, rather than on the C4
aryl moiety (lactam 1), produced nanoparticles (NP2)
that were twofold more active against S. aureus. This
activity was further enhanced by extending the length
of the C3 acrylate tether, as shown for NP3. The in-
crease in biological activity coincided with the relative
levels of bioactivity of the two monomers. Also, NP4,
prepared from diacrylated lactam monomer 4, showed
the same anti-MRSA capabilities as NP2, which was
prepared from the mono-acrylated monomer 2.16 This
indicates that the presence of two acrylate functionalities
on the drug molecule versus only one does not perturb
the biological function or the physical properties of
the resulting nanoparticle.
The emulsions of nanoparticles NP0–4 were evaluated
for antibacterial activity against S. aureus and MRSA
by determining the minimum inhibition concentration
(MIC) values in agar. The monomeric acrylates of these
compounds (1–4) were also examined for comparison.
The MIC values shown in Table 1 represent the lowest
concentration of drug (in lg/mL) needed to completely
inhibit bacterial proliferation. The data revealed promis-
ing MICs for the drug-conjugated emulsions against
both strains of S. aureus, but not for the control (non-
drug-conjugated) nanoparticles (NP0). Thus, only the
emulsified antibiotic-conjugated nanoparticles (NP1–4)
were bioactive while the non-antibiotic-bound nanopar-
In vitro screens determined that these polyacrylate
nanoparticles are non-toxic toward human dermal fibro-
blasts, augmenting the known favorable characteristics
and biocompatibilities of polyacrylate biomaterials.14
Control experiments indicated that the nanoparticle
emulsions were completely stable for at least 24 h at ele-
vated temperatures (up to 60 °C) and in blood serum, as
determined by dynamic light scattering and antibacterial
screening (MIC analysis).17
Figure 2. Atomic force microscopy (AFM) image of NP1 indicates
that the nanoparticles are uniformly spherical and between 30 and
50 nm in diameter.
In conclusion, these polyacrylate nanoparticle antibiot-
ics, or nanobiotics, appear to be an effective enhancer

56
E. Turos et al. / Bioorg. Med. Chem. Lett. 17 (2007) 53–56
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of activity for water-insoluble antibiotics such as N-
methylthio b-lactams. This study revealed several essen-
tial features of polyacrylate nanoparticles that further
illustrate their potential advantages as drug delivery plat-
forms: (1) easy, one-step preparation of antibiotic-conju-
gated polyacrylate frameworks in aqueous media; (2)
facile control of nanoparticle size in the 30–50 nm range;
(3) ability to incorporate water-insoluble drugs directly
onto the polymer framework without the need for
post-synthetic modification of the nanoparticle; (4) en-
hanced anti-MRSA activity of the nanoparticle polymers
versus the non-polymerized form of the drug; and (5)
ability to alter bioactivity of the nanoparticle antibiotic
by changing the location or length of the acrylate linker.
Ongoing studies in our laboratory are focused on the
synthesis and evaluation of nanoparticles for other drug
classes and polymer compositions, and on full character-
ization of their in vitro and in vivo properties.
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Acknowledgments
We thank Dr. Thomas Koob (Shriners Hospital, Tam-
pa) for assistance with cytotoxicity experiments, Gil
Brubaker (University of Florida Particle Engineering
Research Center) for helping with particle analyses,
August Heim for AFM analysis, and Betty Loraam
for help with TEM imaging. Financial support from
the National Institutes of Health (AI05135) is gratefully
acknowledged.
´
K.; Kouri, J. B.; Ramırez, J. T.; Yacaman, M. J.
Nanotechnology 2005, 16, 2346.
14. Odian, G. In Principles of Polymerization, 3rd ed; John
Wiley and Sons, Inc.: New York, 1991.
15. Control experiments indicate that less than 2% of residual
unreacted acrylate was entrapped in the nanoparticle (see
Supporting Information). Moreover, when additional
lactam acrylate was added to the emulsion prior to
evaporation and thin film formation, none of the mono-
mers was incorporated into the polymer matrix and was
able to be recovered quantitatively by extraction of the
film with ethyl ether. This demonstrates that all of the
acrylated lactam monomer was incorporated covalently
into the nanoparticle matrix during emulsion polymeriza-
tion while creating the nanoparticle, and not the result of
further polymerization during the process of evaporation
for NMR analysis.
16. These results suggest that these polyacrylate nanoparticles
are highly crosslinked and that the use of a doubly
acrylated co-monomer such as bis-acrylate 4 does not alter
size, shape, or bioactivity of the nanoparticle. Polyacry-
lates made by emulsion polymerization are known to
possess extremely high molecular weight as a result of
crosslinking via radical backbiting during the polymeriza-
tion process. See the discussions in Ref. 14.
Supplementary data
Synthetic procedures, physical characterization, antibac-
terial and cytotoxicity data for lactams 1–4 and nano-
particles NP0–4. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.bmcl.2006.09.098.
References and notes
1. Zafeiris, S. Nature 2004, 431, 892.
2. Turos, E.; Konaklieva, M. I.; Ren, R. X. F.; Shi, H.;
Gonzalez, J.; Dickey, S.; Lim, D. Tetrahedron 2000, 56,
5571.
17. See Supporting Information.