Colloids and Surfaces A: Physicochem. Eng.

Aspects 370 (2010) 7986

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Colloids and Surfaces A: Physicochemical and
Engineering Aspects
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Enhancing encapsulation efciency of highly water-soluble antibiotic in
poly(lactic-co-glycolic acid) nanoparticles: Modications of standard
nanoparticle preparation methods
Wean Sin Cheow, Kunn Hadinoto

School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637459, Singapore
a r t i c l e i n f o
Article history:
Received 30 June 2010
Received in revised form 11 August 2010
Accepted 30 August 2010
Available online 9 September 2010
Keywords:
Nanoparticle
Encapsulation
Nanoprecipitation
Emulsication
Polymer
Biolm
a b s t r a c t
Effective encapsulations of drugs that are highly soluble in both water and organic solvents are notori-
ously difcult to achieve using standard nanoparticle preparation methods, such as nanoprecipitation
(NPC), single (ESE), and double (DESE) emulsication-solvent-evaporation methods. Modications of the
standard preparation methods are therefore needed to enhance encapsulation efciency of this group of
drugs. The present work investigates the feasibility of enhancing the encapsulation efciency of highly
water and solvent-soluble levooxacin, which is widely used in antibiotic therapy against pulmonary
biolm infections, into poly(lactic-co-glycolic acid) (i.e. PLGA) nanoparticles. In addition, the nanoparti-
cles are evaluated in terms of their drug loading, size, production yield, and in-vitro drug release prole.
Lecithin inclusion into the aqueous phase in the ESE method results in two-fold improvements in the
encapsulation efciency and drug loading (i.e. 23%and 2.3%, w/w, respectively) compared to the standard
method. Similar results are obtained when the water-miscibility level of the oil phase is increased in the
DESE method. Importantly, these nanoparticles possess size 200300nm and biphasic extended drug
release proles suitable for anti-biolmtherapy. Lastly, the nanoparticles can be readily transformed into
inhalable solid dosage forms without jeopardizing their drug loadings and antibacterial activities.
2010 Elsevier B.V. All rights reserved.
1. Introduction
The use of nano-scale formulations inthe deliveryof therapeutic
agents through the pulmonary route has recently gained signi-
cant interest [1]. While nanoparticles can also be delivered through
the oral and parenteral routes, inhalation of nanoparticles repre-
sents a more attractive delivery route, because of the avoidance
of the rst-pass metabolism, ease of administration, and fast drug
absorptions due to the lungs high surface area and thin epithelial
layer. Specically, polymer-based nanoparticle formulations have
been actively researched as inhaled therapeutic carriers owed to
the prospects of having (i) targeted drug deliveries by surface func-
tionalization of the polymer and (ii) sustained drug deliveries by
drug encapsulation in the polymer.
In this regard, antibiotic-loaded polymeric nanoparticles are
highly applicable for the treatment of persistent pulmonary infec-
tions, suchas cystic brosis (CF) andchronic obstructive pulmonary
diseases (COPD), a disease suffered by more than 50 million peo-
ple worldwide [2]. Bacterial biolmcolonies present in the lungs of
CF and COPD patients are surrounded by thick stationary sputum
notorious for binding and deactivating antibiotics, thereby negat-

Corresponding author. Tel.: +65 6514 8381; fax: +65 6794 7553.
E-mail address: kunnong@ntu.edu.sg (K. Hadinoto).
ing the therapeutic effects of inhaled antibiotics. Encapsulation of
antibiotics into polymeric nanoparticles overcomes the problem
of antibiotic deactivations as it prevents interactions between the
antibiotics and the sputum contents.
Even though the encapsulation can also be achieved using
micro-scale polymeric carriers, nano-scale formulations are
favored because the average spacing of sputum mesh is
60300nm[3], such that only nanoparticles can pass through the
sputumtoreachembeddedbiolmcolonies. Byusingnanoparticles
as the carriers, the antibiotics canbe releasedina sustainedmanner
in the vicinity of biolm colonies resulting in enhanced antibacte-
rial efcacies compared to that obtained from antibiotic solutions
[4,5]. In addition, by virtue of their small size, nanoparticles can
effectively evade the pulmonary phagocytic clearance resulting in
an increased antibiotic bioavailability in the lungs [6].
To this end, the nanoparticles are ideally delivered to the lungs
using the dry powder inhaler (DPI) platform. The adoption of the
DPI platformtranslates to portability, improved shelf-life, and high
delivery efciency of the inhaled antibiotics, in contrast to the rel-
ative inconvenience (e.g. lengthy treatment time, non-portability)
and low delivery efciency associated with the conventional neb-
ulization delivery platform [1]. However, nanoparticles in the
dry-powder form exhibit a strong tendency to severely aggregate
due totheir highsurface energy, thus resultinginnon-aerosolizable
powders. Furthermore, nanoparticles, with the exception of par-
0927-7757/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfa.2010.08.050
80 W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986
ticles with aerodynamic diameter (d
A
) <50nm, are predominantly
exhaled fromthe lung without depositing [6]. To effectively deliver
the nanoparticles to the lungs, they are typically transformed into
micro-scale dry-powder aggregates in the inhalable size range
(1d
A
5m) by spray-drying technique [7,8].
To be therapeutically effective, good aerosolization proper-
ties of the nanoparticle aggregates must be complemented by
having a potent antibiotic encapsulatedinto the nanoparticles. Lev-
ooxacin, a uoroquinolone antibiotic, is highly efcacious against
pulmonary biolm cells as it does not have the propensity for spu-
tum binding, unlike aminoglycoside antibiotics [9,10]. However,
the high solubilities of levooxacin in both organic solvents (e.g.
dichloromethane, ethanol, acetone) and water (200300mg/mL
at pH28) render its encapsulation in polymeric nanoparticles
highly inefcient [11].
The low encapsulation efciency of levooxacin is particularly
true when the nanoparticles are prepared by standard methods,
such as nanoprecipitation and emulsication-solvent-evaporation.
For these methods, high drug solubility in either the organic phase
or the aqueous phase, but not both, is required to achieve high drug
encapsulation efciency, as the drug must not leak out from its
initial dissolved phase upon precipitations of the polymer chains
to form the nanoparticles.
In this regard, several investigators have employed standard
nanoparticle preparation methods to encapsulate drugs that are
highly soluble in both water and organic solvents (10
2
mg/mL),
such as procaine hydrochloride, sodiumcromoglycate, and pheno-
barbital, into polymeric nanoparticles [1214]. Not unexpectedly,
low drug encapsulation efciency (<20%, w/w) and low drug load-
ings (1%, w/w) were reported. For this reason, modications to
the standard method were subsequently performed, including (i)
incorporation of salt additives, (ii) pH variation, and (iii) using
alternative solvents, which led to signicant improvements in the
encapsulation efciency (4050%, w/w) and consequently the
drug loading.
The main objective of the present work is to investigate the
effects of modications of the standard nanoparticle preparation
methods on the encapsulation efciency and drug loading of lev-
ooxacin in poly(lactic-co-glycolic acid) nanoparticles (i.e. PLGA).
Threedifferent nanoparticlepreparationmethods (i.e. nanoprecipi-
tation, single, and double emulsication-solvent-evaporations) are
examined. PLGA is selected as the polymeric carrier because of its
well-establishedbiodegradability andbiocompatibility manifested
by its widespread applications as therapeutic carrier materials.
In addition to the encapsulation efciency and drug loading, the
nanoparticles are evaluated in terms of their size, production yield,
and in-vitro drug release prole. In this regard, small nanoparticle
sizes (<300nm) are desirable tofacilitate aneffective sputumpene-
tration, whereas sustained antibiotic release proles are favored as
they canpotentially reduce the dosing frequency andlower the risk
of systemic toxicity. Moreover, the feasibility of transforming the
levooxacin-loaded PLGA nanoparticle suspension into inhalable
dry-powder nanoparticle aggregates by spray drying is investi-
gated. Lastly, the antibacterial efcacy of the solid dosage form
formulation is examined against Pseudomonas aeruginosa (P. aerug-
inosa) biolm cells, which constitute a majority of the bacterial
pathogens present in the lungs of CF and COPD patients [15].
2. Materials and methods
2.1. Materials
PLGA polymer (Purasorb 5004A) is provided by PURAC Bioma-
terials (Netherlands). Chemicals usedinthe nanoparticle synthesis,
i.e. non-hydrochloride formof levooxacin(LEV), dichloromethane
(DCM), acetone, ethyl acetate, Pluronic F-68, poly(vinyl alcohol)
(PVA, MW=1323,000), polyethylene glycol (PEG, MW=4000),
tyloxapol, and chitosan, are purchased from SigmaAldrich (USA).
Drying adjuvants (i.e. lecithin and l-leucine) and biolm growth
medium(i.e. Mueller HintonBrothor MHB) arealsopurchasedfrom
SigmaAldrich (USA). Phosphate buffer saline solution (PBS, pH
7.4) and dialysis membrane with a molecular weight cut-off size of
12,400g/mol used in the in-vitro drug release study are purchased
from 1st Base (Singapore) and SigmaAldrich (USA), respectively.
P. aeruginosa PAO1 strain is obtained from American Type Culture
Collection (ATCC, USA).
2.2. Methods
2.2.1. Nanoparticle preparations by standard methods
2.2.1.1. LEV-loaded PLGA nanoparticles by nanoprecipitation (NPC).
PLGA and LEV are dissolved in water-miscible acetone. Upon addi-
tion of the PLGA/LEV solution into water, the acetone rapidly
diffuses into the aqueous phase resulting in the formation of LEV-
loaded PLGA nanoparticles. Specically, 8mg of LEV and 100mg of
PLGA are dissolved in 5mL of acetone after which the solution is
poured into 10mL aqueous solution of 0.1% (w/v) Pluronic F-68.
The resulting nanoparticle suspension is stirred overnight at room
temperature to evaporate off the acetone present in the aqueous
phase, which is followed by twice centrifugations at 14,000RPM
and two washing cycles to remove the non-encapsulated LEV and
the remaining acetone.
2.2.1.2. LEV-loaded PLGA nanoparticles by single emulsication-
solvent-evaporation (ESE). PLGA and LEV are dissolved in a
water-immiscible solvent (i.e. DCM). Upon addition of the
PLGA/LEV solution into water under ultrasonication, an oil-in-
water nano-emulsion is formed. The DCM is slowly evaporated
to transform the nano-emulsion into a nanoparticle suspension.
Specically, 8mg of LEV and 80mg of PLGA are dissolved in 2mL
of DCM after which the solution is poured into 6mL aqueous solu-
tion of 1.0% (w/v) PVA. The resulting solution is emulsied for 60s
using a Vibra-Cell probe sonicator (VC 5040, Sonics and Materi-
als, USA). The nano-emulsion is next poured into 10mL aqueous
solution of 0.1% (w/v) PVA and the DCM is evaporated overnight at
room temperature. The resulting nanoparticle suspension is cen-
trifuged twice at 11,000RPM and two washing cycles to remove
the non-encapsulated LEV and DCM trace.
2.2.1.3. LEV-loaded PLGA nanoparticles by double-emulsication-
solvent-evaporation (DESE). PLGA and LEV are dissolved separately
in a water-immiscible solvent (i.e. DCM) and deionized water,
respectively. The two solutions are emulsied under ultrasonica-
tion to form w/o nano-emulsion, which is subsequently added to a
second aqueous phase to form w/o/w nano-emulsion. Specically,
9mg of LEV is dissolved in 300L of deionized water and 90mg
of PLGA is dissolved in 3mL of DCM. The aqueous LEV solution
is emulsied in the PLGA organic solution by sonication for 60s.
The resulting nano-emulsion is poured into 12mL aqueous solu-
tion of 2.0% (w/v) PVA and is sonicated for 60s. The nano-emulsion
is stirred overnight at roomtemperature to evaporate off the DCM.
The resulting nanoparticle suspension is centrifuged and washed
following similar procedures described in the ESE method.
2.2.2. Nanoparticle physical characterizations
The drug encapsulation efciency (i.e. % encapsulation) is deter-
mined from the mass ratio of the encapsulated drug to the drug
initially added. The encapsulated drug amount is determined by
subtracting the drug amount present in the supernatant after the
rst centrifugation fromthe drug amount initially added. The drug
W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986 81
concentration in the supernatant is measured using UVvis spec-
trophotometer (UV Mini-1240, Shimadzu, Japan) at absorbance
wavelength of 254nm. The drug loading is determined from the
mass ratio of the encapsulated drug to the total nanoparticles
produced. The nanoparticle production yield is determined from
the mass ratio of the nanoparticle produced to the PLGA initially
added. The nanoparticle size and zeta potential are measured by
photon correlation spectroscopy (PCS) using a Brookhaven 90Plus
Nanoparticle Size Analyzer (Brookhaven Instruments Corporation,
USA).
2.2.3. In-vitro drug release study
Thedrugreleaseratefromthenanoparticles is determinedusing
the dialysis-bagmethodunder a sinkcondition. The dissolutiontest
is conducted at 37

C in a water bath under a continuous stirring.

The dialysis bag containing 2mL nanoparticle suspension is placed
in an opaque bottle to prevent photodegradation of LEV. 6mL PBS
is used as the release medium. At xed intervals, 4mL of the release
medium is withdrawn and the amount of LEV released is mea-
sured by the UVvis spectrophotometer. The withdrawn sample
is replaced with 4mL of fresh PBS to maintain the sink condition.
2.2.4. Spray-drying experiment
The LEV-loaded PLGA nanoparticle suspension is transformed
into dry-powder nanoparticle aggregates using a Bchi B-290
mini spray dryer (Bchi, Switzerland). Drying adjuvants are use
(i) to protect the structural integrity of the nanoparticles upon
drying and (ii) to ensure the nanoparticle aggregates can be recon-
stituted into primary nanoparticles upon their exposure to an
aqueous environment. The particle geometric size (d
G
) and shape
are characterized using a Scanning Electron Microscope (SEM)
model JSM-6700F (JEOL, USA), whereas the particle effective den-
sity (
e
) is determined using a tap densitometer (Quantachromme,
USA). The aerodynamic diameter (d
A
) is determined fromEq. (1) in
which
s
=1g/cm
3
.
d
A
= d
G

S
(1)
2.2.5. Antibacterial activity testing
P. aeruginosabiolmcells arecultivatedaccordingtothemethod
of Harrison et al. [16]. Briey, a bacterial cell suspension used as the
inoculum is adjusted to 1.0 McFarland standard and is diluted by
30-foldinMHBtoproduce1.010
7
colonyformingunits (CFU)/mL.
Next, 150L of the inoculum is transferred to each well of a 96-
well microplate and a 96-peg lid is tted on top of the microplate
to provide a surface for the biolm growth. After 24-h-incubation
in a shaking incubator at 150RPM and 37

C, the peg lid is lifted

and the biolm formed on the pegs is rinsed with PBS to remove
loosely attached planktonic cells.
Table 1
Physical characteristics of nanoparticles prepared by the standard methods.
Run Method Size (nm) % Encapsulation Drug loading (%)
A NPC 80 30 15 0.7
B ESE 190 50 16 1.1
C DESE 190 80 10 1.0
To determine the antibacterial activity, biolm cells formed on
the pegs are exposed to a 20L suspension of the dry-powder
nanoparticle aggregates and 180L of MHB at dosing equal to
7g/mL of LEV. Biolmcells exposed to 20L of PBS and 180L of
MHB are used as the experimental control. After 6, 12, and 24h of
incubation at 37

C, biolm cells remaining on the pegs are rinsed

with PBS and transferred to a newmicroplate containing 200L of
PBS after a ve-minute sonication. The recovered biolm cells are
serially diluted 10-fold and are plated onto agar plates to be incu-
bated overnight at 37

C to determine the viable CFU. The results

reported are based on two replicates. The antibacterial activity of
the nanoparticle suspension is also tested to examine, if any, the
effect of the dry-powder transformation.
3. Results and discussions
3.1. LEV-loaded PLGA nanoparticles prepared by standard
methods
3.1.1. Physical characteristics
A summary of the LEV-loaded PLGA nanoparticles produced
by using the three standard methods (i.e. NPC, ESE, and DESE in
Runs AC, respectively) is presented in Table 1. Despite the similar
initial amount of PLGA used (80100mg), nanoparticles prepared
by the NPC method exhibit a considerably smaller size (100nm)
compared to that of nanoparticles prepared by the ESE and DESE
methods (200nm). The sizes of the three nanoparticles produced
nevertheless remain within the range ideal for sputum penetra-
tions [3].
Particle size distribution of the nanoparticles produced by the
ESE method is presented in Fig. 1A, where a highly uniform distri-
bution is observed. Similarly, uniform size distributions are also
observed for the nanoparticles produced by the NPC and DESE
methods. SEMimage of the nanoparticles inFig. 1Bindicates spher-
ical nanoparticles are produced and veries their highly uniform
size distribution. Zeta potentials of the three nanoparticles are in
the range of () 1030mV indicating colloidally stable nanoparti-
cles.
Inadditionto the smaller size, the NPCmethodresults ina lower
production yield (65%) than the ESE and DESE methods (90%),
which can be attributed to greater particle losses during the cen-
trifugationandwashing steps as a result of the smaller particle size.
Fig. 1. (A) Particle size distribution of LEV-loaded PLGA nanoparticles indicating a highly monodisperse distribution and (B) their SEM image for verication.
82 W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986
Due to the high aqueous solubility of LEV, % encapsulations of LEV
in nanoparticles prepared by the standard methods are expectably
low(16%, w/w) as LEV diffuses out into the aqueous phase during
the nanoparticle preparation steps. Even for the DESE method, the
% encapsulation is also low, despite the double emulsication, due
to the high LEVsolubility in the oil phase, which leads to the escape
of LEV from the inner to the outer aqueous phase.
Consequently, drug loadings of the nanoparticles are also low
(1.1%, w/w), where the NPC method results in the lowest drug
loading at 0.70% (w/w). Experimental uncertainties in the % encap-
sulation and drug loading measurements are between 48% and
5%, respectively, based on three replicates. The extremely lowdrug
loading of the nanoparticles produced by the NPC method is caused
byLEVdiffuses out intotheaqueous phase, together withthewater-
miscible acetone, upon PLGA precipitations, which results in LEV
being predominantly adsorbed onto the nanoparticle surface. Con-
sequently, LEV is washed off more easily during the centrifugation
step, compared to when LEV is encapsulated within the polymer
matrix, resulting in a low drug loading. The dominant presence
of LEV on the surface of the nanoparticles produced by the NPC
method is manifested in its fast release prole as presented in the
next section.
The use of water-immiscible DCM in the ESE and DESE meth-
ods prevents LEV from being predominantly adsorbed onto the
nanoparticle surface as reected in the higher drug loading. In
this regard, the NPC and ESE methods are commonly employed to
encapsulate hydrophobic drugs into nanoparticles, where the lack
of drug solubility in the aqueous phase ensures high encapsulation
efciency. Thus, when highly water-soluble drugs, such as LEV, are
encapsulated using the NPC and ESE methods, % encapsulations
achieved are expectably low.
The DESE method, on the other hand, is typically employed
to encapsulate water-soluble, but not solvent-soluble, drugs, as
the inner aqueous phase acts as a reservoir in which the drug is
dissolved, whereas the oil phase serves as a dissolution barrier pre-
venting the drug leakage fromthe inner tothe outer aqueous phase.
For LEV, which is highly soluble in both the aqueous and oil/organic
phases, standard NPC, ESE, and DESE methods have been shown
to be incapable of producing high % encapsulations. Hence, mod-
ications to the standard methods are needed to enhance the %
encapsulation of LEV.
3.1.2. In-vitro drug release
In-vitro release proles of LEV fromthe nanoparticles produced
in Runs AC are shown in Fig. 2. Experimental uncertainties in the %
drug released, based on three replicates, are approximately 5%. As
previously mentioned, nanoparticles prepared by the NPC method
0
20
40
60
80
100
0 3 6 9 12
%

d
r
u
g

r
e
l
e
a
s
e
d
Time (hours)
Run A
Run B
Run C
Fig. 2. In-vitro drug release proles from nanoparticles prepared by the standard
methods.
exhibit a monophasic burst release prole as a result of LEV being
predominantly present on the nanoparticle surface. Specically,
80% of the loaded LEV is released within the rst hour and the
entire loaded drug is completely released within 3h.
In contrast, nanoparticles prepared by the ESE and DESE meth-
ods display biphasic release proles, where a high burst release
in the rst hour (4050%) is observed due to the presence of
LEV on the nanoparticle surface. The burst release is followed by
a slower release of the remaining LEV contributed by LEV that is
encapsulated within the PLGA matrix. Not all of the loaded LEV,
however, only 8090%, is released after 24h. Nevertheless, the
extended biphasic LEV release proles produced by the ESE and
DESE methods are more desirable than the burst release prole as
they can ensure that LEV concentrations are maintained above the
level needed to inhibit the biolmgrowth in-between dosings [17],
as well as reducing the risk of systemic toxicity.
Thus, nanoparticles prepared by the ESE and DESE methods are
deemed superior compared to those prepared by the NPC method,
because of (i) the higher production yield and drug loading and (ii)
the sustained release proles. For this reason, subsequent modi-
cation attempts to improve the % encapsulation and drug loading
are focused on the ESE and DESE methods. A summary of modica-
tions that are attempted and their corresponding results (i.e. size,
% encapsulation, and drug loading) is presented in Tables 24. The
modications reported in the present work are not aimed towards
Table 2
Physical characteristics of nanoparticles prepared by the modied ESE methods.
Run Modied phase ESE modication Size (nm) % Encapsulation Drug loading (%)
B1
Organic
50% (v/v) DCM:ethyl acetate
200 15 1.0
B2 50% (v/v) DCM:acetone
B3 Aqueous 50% (w/w) PVA:lecithin 24050 23 2.3
Table 3
Physical characteristics of nanoparticles prepared by the modied single-phase DESE methods.
Run Modied phase DESE modication Size (nm) % Encapsulation Drug loading (%)
C1
Inner aqueous
PBS core 190 70 6 0.6
C2 0.1% PEG (w/v) 200 70 8 0.8
C3 0.1% PVA (w/v) 360 120 22 2.1
C4
Oil
50% (v/v) DCM:acetone 110 40 22 2.4
C5 50% (v/v) DCM:ethyl acetate 140 50 20 2.1
W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986 83
Table 4
Physical characteristics of nanoparticles prepared by the modied multiple-phase DESE methods.
Run Inner aqueous phase Oil phase Outer aqueous phase Size (nm) % Encapsulation Drug loading (%)
C6
0.1% (w/v) PVA
DCM:acetone
Standard
170 80 6 0.5
C7 DCM:ethyl acetate 180 80 10 1.0
C8
Standard
50% (w/w) PEG 290 110 11 1.0
C9 50% (w/w) chitosan 720 290 8 0.8
C10 50% (w/w) tyloxapol 720 340 4 0.4
the more usual parameters, suchas stirring speed, pHchange, start-
ing polymer and drug concentrations, or the volume ratio of the
aqueous to organic phase, because these parameters have been
found in our preliminary study to be non-inuential towards the
LEV encapsulation process.
3.2. LEV-loaded PLGA nanoparticles prepared by the modied ESE
methods
Three modications to the standard ESE method are performed,
which are (1, 2) using alternative solvents composed of 50% (v/v)
mixture of DCM and either acetone or ethyl acetate in Runs B1
and B2, and (3) addition of 50% (w/w) lecithin into the aqueous
PVA solution in Run B3. For the effect of using alternative solvents,
precipitations of PLGAchains toformsolidnanoparticles are slower
whencompletelywater-immisciblesolvents, suchas DCM, areused
as it takes longer for the polymer molecules to be exposed to the
aqueous phaseuponemulsications. Theslower PLGAprecipitation
rate provides an opening for the highly water-soluble LEV to leak
out of the organic phase into the aqueous phase resulting inthe low
% encapsulation.
For this reason, varying the water-miscibility level of the solvent
is thought to potentially have an impact on the % encapsulation.
The solvent composition is therefore varied from 100% (v/v) DCM
in the standard ESE method to 50% (v/v) of DCM and ethyl acetate
in Run B1, and to 50% (v/v) of DCM and acetone in Run B2. The
water-miscibility levels of these mixtures increase in the following
order: 100% (v/v) DCM<50% (v/v) DCM/ethyl acetate <50% (v/v)
DCM/acetone, which is attributed to the partial-miscibility of ethyl
acetate and full-miscibility of acetone in water.
Completely replacing DCM with a highly water-miscible sol-
vent, such as acetone, however, is not pursued as it denotes a shift
from the ESE method to the NPC method, which has been found to
produce less-than-ideal nanoparticles. The results of Runs B1 and
B2 in Table 2 nonetheless suggest that the effects of the water-
miscibility level of the solvent on the % encapsulation and drug
loading are minimal, where their respective values remain in the
15% (w/w) and 1.0% (w/w) ranges. Hence, instead of attempting
to reduce the time-scale of the drug leak-out, the next modica-
tion attempt aims to incorporate physicochemical barriers into the
nanoparticles, so that the drug leak-out can be minimized.
In this regard, adding phospholipid lecithin into the aqueous
phase during PLGA nanoparticle preparation has been shown to
result in adsorptions of the amphiphilic lecithin onto the nanopar-
ticle surface through hydrophobic interactions [18]. As LEV is
lipophilic [19], it interacts with the hydrophobic tails of the lecithin
similar to the PLGAlecithin interactions, such that incorporating
lecithin onto the nanoparticle surface can potentially enhance the
% encapsulation by entrapping LEV in the phospholipid layer.
Indeed, two-foldincreases inthe %encapsulationanddrug load-
ing to 23% and 2.3% (w/w), respectively, are observed in Run B3,
where lecithin is added to the 1.0% (w/v) aqueous PVA solution at
50% (w/w) PVA to lecithin ratio. Moreover, in spite of the higher
drug loading, the lecithin inclusion slows down the LEV release
rate due to the hydrophobic interactions as shown in Fig. 3. As a
result, the PLGAlecithin nanoparticles exhibit the ideal extended
biphasic release prole.
3.3. LEV-loaded PLGA nanoparticles prepared by the modied
DESE methods
For the DESE method, the modications are rst performed
one-at-a-time on either the inner aqueous phase or the oil phase
of the w/o/w nano-emulsion to identify formulation parameters
(e.g. solvent, surfactant) that have the most signicant impacts
on the % encapsulation. Subsequently, simultaneous modications
involving the signicant parameters are carried out in both the
inner/outer aqueous and oil phases.
3.3.1. Single-phase modications
First, the effects of modifying the inner aqueous phase on the
% encapsulation and drug loading are investigated by substituting
deionized water used in the standard DESE method with 100% (v/v)
PBS in Run C1, and 0.1% (w/v) aqueous PEG and PVA solutions in
Runs C2 and C3, respectively. The oil and outer aqueous phases
remain unchanged from the standard DESE method.
First, owed to the higher observed solubility of LEV in PBS
than that in deionized water, the use of PBS as the inner aque-
ous phase is thought to be able to increase the % encapsulation,
where LEV would remain longer in the aqueous core upon emul-
sications due to the lower degree of supersaturation. However,
the results of Run C1 presented in Table 3 suggest otherwise,
where the % encapsulation and drug loading are lowered to 6% and
0.6% (w/w), respectively, from 10% and 1.0% (w/w) in the standard
DESE method. The nanoparticle size is nevertheless unaffected and
remains in the 200300nm range.
Second, the inclusion of a surfactant in the inner aqueous phase
of the DESE method has been shown at the micro-scale to enhance
the primary w/o emulsion stability, which increases the aqueous
pore volume of the carrier particles, resulting in higher % encap-
sulation and drug loading [20]. For this reason, the inclusion of
surfactants in the aqueous core is nowattempted at the nano-scale
0
20
40
60
80
0 6 12 18 24
%

d
r
u
g

r
e
l
e
a
s
e
d
Time (hours)
Run B w/o lecithin
Run B3 w/ lecithin
Fig. 3. Effect of lecithin inclusion on the in-vitro drug release prole.
84 W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986
to examine whether a similar nding is observed. Using PEG as
the surfactant, the results of Run C2 indicate that % encapsulation
and drug loading are lowered to 8% and 0.8% (w/w), respectively,
without any signicant impacts on the nanoparticle size.
Incontrast, usingPVAas thesurfactant inRunC3yields nanopar-
ticles having higher % encapsulation and drug loading (i.e. 22% and
2.1%, w/w, respectively), which is similar to the results obtained
with the lecithin inclusion in the ESE method (i.e. Run B3). How-
ever, the nanoparticle size in Run C3 is increased to 400500nm
range, which makes themless suitable for sputumpenetration. The
signicant difference in the effects of PEGand PVA inclusion on the
%encapsulationis likelydue tothe fact that adsorbedPEGis washed
off the PLGA nanoparticle surface more easily than PVA [21]. Sig-
nicantly, the results of Runs B3 and C3 indicate that the presence
of surfactant layer on the PLGA nanoparticle surface can enhance
the % encapsulation and drug loading provided that the surfactant
remains adsorbed upon washing.
Third, the modications are performed in the oil phase by
replacing the 100% (v/v) DCM in the standard DESE method with
50%(v/v) mixture of DCM/acetone inRunC4andDCM/ethyl acetate
in Run C5. DCMcannot be completely replaced as the DESE method
requires the presence of a completely water-immiscible solvent.
In this regard, the type of solvent used as the oil phase has been
reported to impact the % drug encapsulation in nanoparticles due
to the different nanoparticle precipitation rates depending on the
water-miscibility level of the solvent used [22].
Specically, in the second emulsication step, the polymer pre-
cipitates at a rate proportional to the water-miscibility level of the
solvent, where the more water-miscible the solvent is, the faster
the polymer precipitates to form w/o/w nano-emulsion as a result
of the faster exposure to the aqueous phase. The faster the poly-
mer precipitates andencapsulates the drug inthe process, less drug
leaks out fromtheinner totheouter aqueous phase, whichintheory
should improve the % encapsulation.
For this reason, the effect of varying the water-miscibility level
of the oil phase is investigatedinRuns C4 andC5 by adding miscible
acetone and partially miscible ethyl acetate into the DCM. In con-
trast to the results of a similar study in the ESE method (i.e. Runs
B1B2), the results in Table 3 indicate that increasing the water-
miscibility level of the oil phase does lead to improvements in the
% encapsulation and drug loading to 2022% and 2.12.4% (w/w),
respectively, which is more than two-fold higher than the values
obtained from the standard DESE method.
The slightly higher drug loading achieved in Run C4 is attributed
to the higher water-miscibility of the DCM/acetone mixture com-
pared to the DCM/ethyl acetate mixture. Importantly, the higher
drug loading does not jeopardize the nanoparticle sizes, which in
fact are slightly reduced to be below200nm. Despite the increased
drug loading, the in-vitro LEV release proles of Runs C4 and C5
presented in Fig. 4 are similar to that of the standard DESE method
in Run C, where 90% of the drug is released within 6h.
The difference in the effects of varying the water-miscibility
level on the % encapsulation between the ESE and DESE methods
is postulated to be caused by the fact that in the DESE method, the
drug is dissolved in the inner aqueous phase, such that faster poly-
mer precipitations at higher water-miscibility levels can prevent
the drug leak-out from the inner aqueous core to the outer aque-
ous phase resulting in higher % encapsulation. In contrast, the drug
is dissolved in the oil phase (i.e. solvent phase) in the ESE method,
such that even though faster polymer precipitations increase the
% encapsulation, the diffusion of the water-miscible component of
the oil phase (i.e. either ethyl acetate or acetone) into the aqueous
phase uponemulsicationresults inthe dissolveddrugbeingtrans-
ported out to the aqueous phase together with the water-miscible
solvent. As a result, the increase in the % encapsulation owed to the
faster polymer precipitation is cancelled out by the drug loss due to
0
20
40
60
80
100
0 6 12 18 24
%

d
r
u
g

r
e
l
e
a
s
e
d
Time (hours)
Run C
Run C4
Run C5
Fig. 4. Effect of varying the water-miscibility level of the oil phase on the in-vitro
drug release prole.
the solvent outward diffusion resulting in insignicant net effects
on the % encapsulation.
3.3.2. Multiple-phase modications
To examine whether further improvements in the % encapsula-
tion and drug loading can be achieved, simultaneous modications
involving the inner and outer aqueous phases, as well as the oil
phase, are investigated in Runs C6C10 presented in Table 4. 0.1%
(w/v) aqueous PVA solution is used as the inner aqueous phase in
Runs C6C10as it has beenfoundinRunC3to signicantly improve
the % encapsulation and drug loading, though it also results in a
larger nanoparticle size.
First, the effects of simultaneously modifying the inner aqueous
phase and the oil phase, while keeping the outer aqueous phase
unchanged (i.e. 2.0% (w/v) aqueous PVA solution), are examined
in Runs C6 and C7. DCM/acetone and DCM/ethyl acetate mixtures
are used as the oil phase in Runs C6 and C7, respectively, as they
have beenfoundtoincrease the %encapsulationinRuns C4C5. The
results, however, indicate that a synergistic effect is not observed
whentheinner aqueous phaseandtheoil phasearemodiedsimul-
taneously.
In fact, less favorable results in terms of the % encapsulation and
drug loading are observed in Runs C6C7 compared to the single-
phase modication results. Specically, the % encapsulation is back
to be 10% (w/w) similar to that obtained using the standard DESE
method. Similarly, the drug loading falls below 1.0% (w/w) and the
nanoparticle size is back in the 200300nm range.
Second, the effects of simultaneously modifying the inner
and outer aqueous phases, while keeping the oil phase remains
unchanged (i.e. 100% (v/v) DCM), are investigated in Runs C8C10.
The outer aqueous phase is modied by adding new surfactants or
polymers widely used in drug delivery formulation, such as PEG,
chitosan, and tyloxapol, into the 2.0% (w/v) aqueous PVA solution.
The presence of these materials at 50% (w/w) ratio in the outer
aqueous phase is thought to have the potential to inuence the
drug leak-out rate.
The results nonetheless indicate that the % encapsulation, drug
loading, and nanoparticle size deteriorate signicantly compared
to the single-phase modications in Runs C3C5. Specically, the
% encapsulations are low between 4% and 11% (w/w) resulting
in low drug loadings between 0.1% and 1.0% (w/w). Furthermore,
the nanoparticle sizes in Runs C9 and C10 are excessively large at
700nm, hence signifying the importance of either PVA or PEG
W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986 85
in maintaining the colloidal stability of the w/o/w nano-emulsion
formed.
To summarize, for the DESE method, simultaneous mod-
ications involving multiple phases negatively affect the %
encapsulation and drug loading. In contrast, single-phase modi-
cations in Runs C3C5 result in two-fold improvements in the %
encapsulation and drug loading. However, the large nanoparticle
size produced in Run C3 (i.e. 400nm) is undesirable due to the
lack of sputumpenetration ability. For the ESE method, the lecithin
inclusion in Run B3 similarly results in a two-fold improvement
in the % encapsulation and drug loading, while maintaining the
nanoparticle size in the 200300nm range.
Importantly, the nanoparticles produced in Run B3 exhibit a
slower sustained LEV release prole, which make them more
attractive than those produced in Runs C4 and C5, despite the sim-
ilarities in terms of their size, % encapsulation, drug loading, and
production yield. In addition, the ESE method is simpler than the
DESE method owed to the single-emulsication step. For this rea-
son, the lecithin-modied nanoparticles produced in Run B3 are
selected as the optimal formulation for further spray-drying and
antibacterial activity testing experiments.
3.4. Spray-drying production of dry-powder LEV-loaded PLGA
nanoparticles
The following spray-drying conditions are employed, i.e.
inlet temperature =100

C, feed rate =2.8mL/min, atomizing ow

rate =350L/h, and feed concentration=1.0% (w/w). Due to the high
drying temperature, which is above the glass transition tempera-
ture of PLGA(i.e. 35

C), the inclusion of leucine and PVAas drying

adjuvants is necessarytoprotect thestructural integrityof thePLGA
nanoparticles. The ratio of the nanoparticles to the drying adju-
vants is maintained at 1:1. Dry-powder nanoparticle aggregates
produced at this spray drying condition possess d
G
between 6 and
9mand low
eff
(0.3g/cm
3
) owed to the hollowmorphology as
shown in Fig. 5A. Due to the hollow morphology, d
A
of these par-
ticles is between 3 and 5m rendering them suitable for inhaled
delivery.
Importantly, the drug loading of the dry-powder nanoparticles
is similar in magnitude to that of the suspension form indi-
cating a minimal drug loss in the drying process. Furthermore,
these nanoparticle aggregates readily reconstitute into individual
nanoparticles upon their exposure to an aqueous environment. The
nanoparticle size after the re-constitution in Fig. 5B, however, is
about twice larger than their size prior to the dry-powder transfor-
mation, which is due to the coalescence of the PLGA nanoparticles
upon exposure to a high temperature above their glass transition
temperature as can be seen in Fig. 5C.
3.5. Antibacterial activity of dry-powder LEV-loaded PLGA
nanoparticles
To ensure that the activity of the nanoparticles is not affected
by their transformation into the dry-powder form, antibacterial
activities of both the dry-powder and suspension forms of the
nanoparticles are tested against P. aeruginosa biolm cells. The
antibacterial activities of the nanoparticles are evaluated in a time-
kill study after 6, 12, and 24h of antibiotic exposures using a LEV
dose of 70.3g/mL, which is above the minimuminhibitory con-
centration (MIC) of the planktonic cells (2g/mL) as biolmcells
are well-known to exhibit a higher antibiotic tolerances than their
planktonic cell counterparts.
The experimental control (i.e. biolm cells exposed to PBS)
shown in Fig. 6A remains at a density of 8log (CFU/peg) through-
out the duration of the study signifying a steady-state biolm
density when no antibiotic is introduced. After 6h of exposure to
Fig. 5. (A) SEM image of dry-powder nanoparticle aggregates prepared by spray-
drying, (B) particle size distribution of the nanoparticles before the dry-powder
transformation and after re-constitution, and (C) SEM image of the re-dispersed
nanoparticles.
the nanoparticles, 99.999% of the biolm cells are killed with only
3log (CFU/peg) of surviving biolm cells, which is attributed to
the high antibiotic concentration produced by the burst release.
The number of biolm cells, however, increases at the 12th and
24th hour to 5log (CFU/peg) as the surviving biolm cells begin to
multiply due to the subsequent slower antibiotic release. Overall,
99.9%of thebiolmcells areeradicatedafter 24h. Importantly, at all
time-points, the extents of biolmcell eradications afforded by the
dry-powder form are nearly identical to that of the nanoparticle
suspension, which indicates that the dry-powder transformation
does not affect the antibacterial activity of the nanoparticles.
In addition, to verify that the biolm eradications presented in
Fig. 6A are solely attributed to the LEV-loaded nanoparticles, and
not because of the presence of hazardous solvent trace from the
nanoparticle preparation steps, antibacterial activity of the blank
nanoparticles (i.e. no antibiotic) is examined. The results in Fig. 6B
indicate that the blank nanoparticles, in their suspension and dry-
86 W.S. Cheow, K. Hadinoto / Colloids and Surfaces A: Physicochem. Eng. Aspects 370 (2010) 7986
Fig. 6. (A) Antibacterial activities of the LEV-loaded nanoparticles at three differ-
ent time-points (DP3=dry-powder nanoparticles, NP3=nanoparticle suspension)
against P. aeruginosa biolm cells and (B) non-activity of blank nanoparticles.
powder forms, do not exhibit any antibacterial activities towards
the biolm cells as the log (CFU/peg) of the biolm cells exposed
to the blank nanoparticles are similar in magnitude to that of the
control experiment.
4. Conclusions
LEV-loaded PLGA nanoparticles are prepared by employing the
standard and modied NPC, ESE and DESE methods. In compari-
son to the standard methods, single-phase modications involving
(i) the addition of lecithin into the aqueous phase (ESE) and (ii)
the use of alternative solvents (DESE) result in two-fold improve-
ments in the % encapsulation and drug loading (i.e. 2224% and
2.12.4%, w/w, respectively). Signicantly, theexperimental results
indicatethat simultaneous modications involvingmultiplephases
do not lead to additive effects of the single-phase modications. In
other words, an observed doubling of the % encapsulation when
two single-phase modications are performed separately does not
translate to quadrupled % encapsulation when the two modi-
cations are performed simultaneously. The optimal formulation,
which is evaluated in terms of the nanoparticle size, % encapsu-
lation, drug loading, in-vitro drug release prole, and production
yield, has been identied in the form of PLGA-lecithin nanopar-
ticles prepared by the ESE method. Lastly, the nanoparticles can
be straightforwardly transformed into the inhalable solid dosage
form by spray-drying with the inclusion of drying adjuvants. The
dry-powder nanoparticles exhibit similar antibacterial activities as
their aqueous suspension form against P. aeruginosa biolm cells.
Acknowledgement
A nancial support from Nanyang Technological Universitys
Start-Up Grant (Grant No. SUG 8/07) is gratefully acknowledged.
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