Journal of Microencapsulation, 2011; 28(5): 430441

2011 Informa UK Ltd.

ISSN 0265-2048 print/ISSN 1464-5246 online
DOI: 10.3109/02652048.2011.576786

Insulin-loaded PLGA microparticles: flow focusing versus double

emulsion/solvent evaporation
M. J. Cozar-Bernal1, M. A. Holgado1, J. L. Arias2, I. Munoz-Rubio1, L. Martn-Banderas1,
J. Alvarez-Fuentes1 and M. Fernandez-Arevalo1
1
Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Seville, Seville, Spain,
and 2Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada,
Granada, Spain

Abstract
Context: Oral administration of insulin is severely limited by very low bioavailability. Biocompatible poly-
meric nanocarriers have been investigated to overcome this problem. Flow focusing (FF) has revolutionized
current engineering of poly(D,L-lactide-co-glycolide) (PLGA) based micromedicines. This technique has
never been used to formulate insulin-loaded PLGA microparticles.
Objective: Investigation of the benefits rising from the synthesis of insulin-loaded PLGA microplatforms by
FF, compared to double emulsion/solvent evaporation method.
Materials and methods: Both synthesis methodologies were compared in terms of geometry, surface phys-
For personal use only.

icochemical properties and insulin vehiculization capabilities. The stability of the peptide during the for-
mulation procedure was further analysed.
Results: FF permitted the preparation of insulin-loaded microcarriers with better geometry and physico-
chemical properties for the oral route, along with greater insulin loading capabilities and sustained insulin
release kinetics.
Discussion and conclusion: Results have lead to the identification of the best formulation conditions for the
engineering of insulin-loaded PLGA microparticles against diabetes.
Keywords: Double emulsion/solvent evaporation, electrokinetic properties, flow focusing, insulin,
poly(D,L-lactide-co-glycolide), surface thermodynamics

Introduction administration of insulin generally leads to an important

Insulin is widely used to maintain normal glycaemia levels
in patients suffering from type I diabetes mellitus (DM),
and in an important percentage of patients suffering from
type II DM. This biomacromolecule is made of two poly-
peptide chains, which have been extensively characterized
in terms of chemical structure, stability and activity (Zhou
and Po, 1991a, 1991b; Matsuura et al., 1993). The therapeu-
tic effect of exogenous insulin relies on its capability to
determine glucose cellular uptake (mainly in muscle and
adipose tissue), DNA replication and protein synthesis, and
the activity of several enzymatic systems (e.g. leading to
controlled hepatic gluconeogenesis). The subcutaneous
patient discomfort, non-compliance and fluctuations in
glycaemia levels. As an alternative, the oral route has
faced important limitations determining a very low bio-
availability, i.e. self-aggregation problems, susceptibility
to acid pHs and proteases naturally occurring in the gas-
trointestinal tract, and very poor absorption through the
intestinal mucosa (a consequence of its large size and
hydrophilicity; Zhou and Po, 1991b).
Significant research efforts have been focused on the
improvement of the oral bioavailability of insulin,
e.g. by the introduction of chemical modifications in the
insulin structure, and the co-administration of absorption
enhancers or protease inhibitors, to cite just a few

Address for correspondence: M. J. Cozar-Bernal, Departamento de Farmacia y Tecnologa Farmaceutica, Facultad de Farmacia, Universidad de Sevilla,
Seville, Spain. Tel: 34 954 551 624. Fax: 34 954 556 085. E-mail: cozar@us.es

(Received 28 Nov 2010; accepted 28 Mar 2011)

http://www.informahealthcare.com/mnc
430

(Myers et al., 1997; Bernkop-Schnurch, 1998). More
recently, the formulation of biocompatible polymeric
microplatforms has been proposed as a revolutionary strat-
egy to improve the protection of the biomacromolecule
from in vitro and in vivo degradation, to enhance its oral
absorption and to control the insulin release kinetics
(Bhardwaj et al., 2005; Cui et al., 2006; Hamishehkar
et al., 2009; Sonaje et al., 2010). Moreover, the possibility
of circumventing the first pass metabolism of insulin by
using such microparticulate system has been described.
This has been hypothesized to be the consequence of
oral absorption processes associated to microparticle
uptake by Peyers patches through receptor mediated
endocytosis (lymphatic uptake), and/or by paracellular
pathways (non-lymphatic uptake; Norris et al., 1998).
Poly(D,L-lactide-co-glycolide) (PLGA) has been widely
used in the formulation of drug micro- and nano-carriers,
thanks to its excellent biocompatibility and biodegradabil-
ity (Kumar et al., 2001; Holgado et al., 2008; Santander-
Ortega et al., 2009). Unfortunately, despite the wide variety
of synthesis procedures proposed for the formulation of
insulin-loaded PLGA particles, the hydrophilic character
of the peptide determines a thermodynamically unfa-
voured loading into such a hydrophobic polymer
(Mundargi et al., 2008). An alternative synthesis technique
for the incorporation of biomacromolecules (peptides, pro-
For personal use only.
teins) and drug molecules into PLGA particles has been
lately developed: flow focusing (FF) methodology
(Martn-Banderas et al., 2005; Holgado et al., 2008, 2009).
Advantageously, the technique offers very interesting
possibilities over classical synthesis procedures (i.e.
double emulsion/solvent evaporation (DE/SEV), solvent
extraction/evaporation) in the engineering of PLGA
particles for oral insulin delivery. In detail, this technology
is expected to present a controlled particle geometry (per-
mitting narrow size distributions), high loading values,
physical and chemical compatibility, exceptional perfor-
mance and large-scale production (Martn-Banderas et
al., 2006; Holgado et al., 2009). The methodology involves
the combination of hydrodynamic forces with a specific
geometry (Figure 1; Ganan-Calvo, 1998).
In this work, we describe the formulation of insulin-
loaded PLGA microparticles for the oral route. To that
aim, two synthesis methodologies have been investigated:
the traditional DE/SEV method, and the novel FF tech-
nique. A comparative study between both procedures
was undertaken to define their influence on the geometry
(size and shape), electrophoretic properties, surface ther-
modynamics and insulin vehiculization characteristics
(loading and release) of PLGA particles.
Materials and methods
Materials
Insulin human recombinant, Resomer RG 502 (PLGA
50:50, Mw: 12 000 Da; inherent viscosity: 0.24 dL/g), poly-
vinyl alcohol (PVA, Mw: 16 000 Da), and trifluoroacetic acid
Insulin-loaded PLGA microparticles 431
(TFA) were purchased from SigmaAldrich (Germany),
Boehringer Ingelheim (Germany), Fluka (Germany) and
Merck (Germany), respectively. All other chemicals were
of analytical quality from Panreac (Spain). Water used in
the experiments was deionized and filtered (Milli-Q
Academic, Millipore, France).
Reversed phase-high performance liquid chromatogra-
phy (RP-HPLC) was validated and verified for accuracy,
precision and linearity in all the experimental conditions
investigated. Briefly, RP-HPLC was done in a Hitachi
LaChrom (D-7000) Series HPLC system equipped with a
L-7200 automatic injector, an interphase D-7000 and a
quaternary pump (model L-7100), coupled with a L-7455
diode array detector (Merck-Hitachi, Darmstadt,
Germany). A Zorbax SB-C18 column (5 mm, 4.6  250 mm,
USA) kept at 25.0  0.1 C (L-2350 column oven, Elite
LaChrom ) was used in this analysis. The mobile phase
consisted of an acetonitrile solution (solution A), and an
aqueous 0.1% (v/v) TFA (solution B) that was filtered
through a 0.22 mm nitrocellulose membrane filter
(Millipore, Madrid, Spain), and degassed under vacuum.
The linear gradient of the mobile phase (solution
A:solution B) was subsequently kept to 30:70, 40:60 and
70:30 from t 05 min, 610 min and 1115 min, respec-
tively; 50 mL of the eluent were pumped at a flow rate of
1000 mL/min. Insulin was monitored at a wavelength of
214 nm. All the measurements were done in triplicate at
room temperature, and the total peak area was used to
quantify the peptide. Data collection and calculation
were carried out by using HSM software (Merck-Hitachi,
Darmstadt, Germany).
Methods
Preparation of insulin-loaded PLGA microparticles
PLGA microparticles with a theoretical insulin loading up
to 4.76% (w/w) were prepared either by water-in-oil-in-
water (w/o/w) DE/SEV, or FF. In the former case, an aque-
ous solution of insulin [pH  2.3; containing acetic acid
(10%, v/v) and Tween 80 (5%, v/v)] was added to 1 mL
of a PLGA solution in ethyl acetate, under sonication for
5 min at 50 kHz (sonic power: 50 W; Selecta S.A., Barcelona,
Spain). The resulting w/o emulsion was poured into 0.3%
(w/v) PVA aqueous solution, and sonicated for 15 min. As a
result, a w/o/w emulsion was formed, which was then
diluted in 20 mL of a 2% (w/v) PVA aqueous solution.
Finally, mechanical stirring at 300 rpm and at 25.0  0.5 C
was continued for 3 h (magnetic stirrer hotplate SM6,
Jepson Bolton, Hertfordshire, UK) to completely evaporate
the organic solvent and to obtain the solid microparticles.
The synthesis of insulin-loaded PLGA microparticles by
FF involves the preparation of a w/o emulsion by using a
standard FF nozzle in a liquidliquid configuration
(Figure 1; Holgado et al., 2009). Briefly, the w/o emulsion
was obtained by mixing under sonication at 50 kHz (sonic
power: 50 W; Selecta S.A., Spain) for 5 min an aqueous solu-
tion of insulin (10 mg/mL) [pH  2.3; containing acetic acid
(10%, v/v) and Tween 80 (25%, v/v)] with a PLGA solution
 432 M. J. Cozar-Bernal et al.

Figure 1. FF device used in the formulation of polymeric microparticles. It consists of a pressurized chamber with a continuous focusing fluid supply.
Inside, a hydrodynamic funnel shaped lens is generated when the flowing focusing fluid (1) undergoes a pressure drop through an orifice. By feeding an
immiscible liquid flow (focused fluid, 2) into this hydrodynamic funnel, a steady thin liquid jet is created in the core of the co-flowing focusing stream. As a
consequence, the created micro- or nano-liquid jet leaves the chamber through the orifice (meniscus, 3), along with the focusing fluid. Liquid jet diameter
is much smaller than the orifice diameter, thus precluding any contact. Finally, capillary instability breaks up the stationary liquid jet into homogeneously
For personal use only.

sized droplets.

in ethyl acetate (1%, w/v). The emulsion was sprayed by
using a standard FF nozzle (model Avant 2 (D 50 mm),
Ingeniatrics Tecnologas S.L., Sevilla, Spain) immersed
into the final continuous phase. The disperse phase was
injected through a capillary tube using a syringe pump.
Then it was focused and pressed out of the device through
the orifice (50 mm), using water or phosphate buffered
saline (PBS; pH 7.4  0.1, or 9.0  0.1) as focusing fluid.
The focusing fluid was injected through a capillary tube
using a HPLC pump. The ionic strength of the focusing
fluid was adjusted to 50 or 500 mS/cm to increase the insu-
lin content. Oil drops were generated inside a 1% (w/v)
PVA solution without any shape deformation. The final
PVA concentration of the continuous phase was 0.25%
(w/v). The resulting w/o/w emulsion was continuously stir-
red (magnetic stirrer hotplate SM6, Jepson Bolton, UK) at
25.0  0.5 C for 3 h to completely evaporate the organic
solvent, and the PLGA microparticles were subsequently
obtained.
In both the synthesis methodologies, the microparticle
suspension was finally subjected to a cleaning procedure
that involved three cycles of centrifugation (20 min,
10 000 rpm; Centrifuge eppendorf 5804R by Eppendorf,
Germany) and redispersion in 10 mL water. Polymeric
microparticles were then frozen in liquid nitrogen and
lyophilized at 80.0  0.5 C and 0.05 mbar during 24 h
(Freeze Dryers Telstar Cryodos by Telstar, Spain). The
lyophilized microparticles were stored at 4.0  0.5 C until
their use. All the formulations were prepared in triplicate.
Table 1 summarized the formulation conditions followed
to prepare insulin-loaded PLGA microparticles by DE/SEV
and FF.
Characterization of insulin-loaded PLGA microparticles
The geometry (size and morphology) of insulin-loaded
PLGA microparticles was determined from scanning elec-
tron microphotographs (Philips XL-30, Philips Electron
Optics, Eindhoven, The Netherlands). Prior to observation,
a dilute microparticle suspension (0.1%, w/v) was soni-
cated for 5 min, and drops were placed on copper grids
with formvar film. The grids were then dried at
35.0  0.5 C in a convection oven. Additionally, mean par-
ticle size and particle size distributions were measured at
25.0  0.5 C by laser diffraction (Partica LA-950V2, Horiba
laser diffraction particle size analyser, Kyoto, Japan).
Particle measurement ranged from 0.01 to 3000 mm; parti-
cle size distribution was determined by following the Mie
theory for scattering.
Surface electrical properties of PLGA microparticles
were characterized by electrophoretic mobility (ue) deter-
minations (Malvern Zetasizer 2000 electrophoresis device,
Malvern Instruments Ltd., Malvern, UK). The influence of
pH and ionic strength on ue values of all the formulations
was also evaluated. To that aim, PLGA aqueous suspen-
sions (0.1%, w/v) were prepared by adjusting KNO3
concentration, and pH (with KOH or HNO3), respectively.
Measurements were done at 25.0  0.5 C, after 24 h of
contact at this temperature under mechanical stirring
 Insulin-loaded PLGA microparticles 433

Table 1. Formulation conditions in the preparation of insulin-loaded PLGA microparticles by DE/SEV and FF methodologies.

Formulation Insulin (%, w/v) PLGA (%, w/v) Focusing fluid Ionic strength (mS/cm) PVA solution (pH)

DE/SEV-1 0.5 10 6.5

DE/SEV-2 0.5 10 9.0
DE/SEV-3 1 1 6.5
DE/SEV-4 1 1 9.0
FF-1 1 1 water 50 6.5
FF-2 1 1 water 50 9.0
FF-3 1 1 water 500 6.5
FF-4 1 1 water 500 9.0
FF-5 1 1 pH 7.4 50 6.5
FF-6 1 1 pH 7.4 50 9.0
FF-7 1 1 pH 7.4 500 6.5
FF-8 1 1 pH 7.4 500 9.0
FF-9 1 1 pH 9.0 50 6.5
FF-10 1 1 pH 9.0 50 9.0
FF-11 1 1 pH 9.0 500 6.5
FF-12 1 1 pH 9.0 500 9.0

(50 rpm). The experimental uncertainty of the measure-
ments was always below 5%. The theory of OBrien and
White (1978) was used to transform ue data into zeta poten-
tial () values. The electrokinetic characterization of the
formulations further allowed to: (1) clarify the influence
of the synthesis techniques on the surface electrical prop-
erties of PLGA microparticles and (2) characterize the type
For personal use only.
Evaluation of insulin loading to PLGA microparticles
Samples of insulin-loaded PLGA microparticles (20 mg)
were added to 100 mL of acetonitrile, and the mixture was
vortexed for 5 min; 1.4 mL of a 0.01 M HCl solution was
subsequently added to that sample solution and then vor-
texed for 2 min. Upon centrifugation of the final sample
solution (5000 rpm, 4.0  0.5 C, 20 min) the supernatant

of insulin loading to PLGA (surface adsorption onto the
microparticle surface or absorption into the polymeric
matrix; Arias et al., 2007, 2010; Holgado et al., 2009).
Surface thermodynamics of PLGA microparticles were
investigated by atomic force microscopy (AFM; Vadillo-
Rodriguez et al., 2005). The evaluation of the hydrophobic
character of the microparticles was carried out by using
polystyrene (a more hydrophobic material) as reference.
Samples were prepared by incorporating a drop of PLGA
suspension in water, and subsequently drying the poly-
meric dilution at room temperature on properly cleavaged
mica sheets. AFM measurements were conducted in tripli-
cate under atmospheric conditions by using a molecular
imaging PicoPlus atomic force microscope (Pico Scan 2500,
Agilent Technologies, Santa Clara, CA). AFM images were
performed in contact mode (to evaluate the parameters
associated to hydrophobicity: contrast of friction and con-
tact curves) by using an atomic force ranging from 1 to
10 nN on gold-coated triangular silicon nitride tips
mounted on microfabricated cantilevers (spring constant:
0.58 nN/nm). Height and surface roughness were
determined by image analysis (PicoScanTM 5.3.3 image
analysis software, Molecular Imaging Corporation, Los
Angeles, CA). Transformation of voltage values into
cantilever deflection (dc) data was possible from the
slopes of the retraction force (F)-distance curve in the
region where probe and sample were in deep contact.
The dc data were then used to estimate the interaction
forces with hydrophobic surfaces. Johnson, Kendall and
Roberts (JKR) theory was used to establish a direct
relationship between the adhesion force and the surface
free energy of interaction of the microparticles
(Johnson et al., 1971).
was filtered through a 0.45 mm nylon membrane filter
(Millipore, Spain) before RP-HPLC analysis (Kumar et al.,
2007; Hamishehkar et al., 2009). Insulin loading to PLGA
microparticles was analysed at 214 nm, and indicated in
terms of insulin entrapment efficiency (EE, %) [(incorpo-
rated insulin (mg)/total insulin in the colloidal suspension
(mg))  100] and insulin loading (%) [(incorporated insulin
(mg)/PLGA microparticles (mg))  100]. The normal dis-
tribution of each continuous variable was assessed using
the ShapiroWilk test. Factorial analysis of the variance was
undertaken to determine the influence on EE of: (1) the
amount of PLGA, and the pH of the PVA solution when
DE/SEV was the method followed in the formulation of
the microparticles and (2) the type of focusing fluid, envi-
ronmental ionic strength and pH of the PVA solution when
FF was the technique followed in the formulation of the
microcarrier. In this way, a preliminary investigation by
circular dichroism (CD) was conducted to clarify the influ-
ence of pH on the stability of insulin. It was determined
that the peptide was very stable in alkaline environments
(data not shown).
The vehiculization of insulin into PLGA microparticles
was also qualitatively characterized in triplicate by CD and
fluorescence spectroscopy (Bloemendal et al., 2005). These
experimental techniques further permitted to evaluate the
integrity and stability of the peptide upon loading into the
polymeric particles, by analysing its secondary and tertiary
structures, respectively (Tatford et al., 2004; Shelma et al.,
2010). For instance, changes in the secondary and tertiary
structures of insulin can be detected by far-UV (200
260 nm) and near-UV (250320 nm) CD analysis, respec-
tively. Jasco CD-2095 CD detector (Jasco, Tokyo, Japan)
was used for on-line monitoring of CD of the samples
 434 M. J. Cozar-Bernal et al.
in the UV wavelength range (220420 nm). Fluorescence
determinations (F-2500 fluorospectrophotometer, Hitachi,
Schaumburg, IL) were carried out at an excitation wave-
length of 275 nm, and emission intensities were scanned
from 290 to 450 nm (Nyambura et al., 2009). Fluorescence
spectrum of the biomacromolecule will depend on the
tyrosine residues (A14, A19, B16 and B26) and their chem-
ical environment. The splits of excitation and emission
were 10 nm (Yong et al., 2009). Before analysis, sample
solutions were appropriately diluted with a 0.01 M HCl
solution to assure absorbance values 50.1 at 280 nm.
The spectra were normalized for concentration differences.
Finally, all samples for both analytical techniques were pre-
pared by dissolving insulin, blank (non-loaded with insu-
lin) PLGA, a physical mixture of insulin and PLGA, and
insulin-loaded PLGA in 0.01 M HCl, and contained a final
insulin concentration of  0.1 mg/mL.
In vitro insulin release from PLGA particles
Insulin release from PLGA microparticles was determined
in triplicate following the dialysis bag method. PBS
(pH 7.4  0.1) was used as the release medium. The dial-
ysis bag with a cutoff of 12 00014 000 Da (Spectrum
Spectra/Por 6 dialysis membrane tubing, Spectrum
Laboratories, USA) retained the PLGA microparticles and
allowed the free insulin to diffuse into the dissolution
For personal use only.
media. These bags were soaked in PBS during 2 h before
use. Then, 1 mL of an insulin-loaded PLGA microparticle
suspension (containing 20 mg of microparticles) was
poured into a dialysis bag with the two ends fixed by
clamps. The bags were then placed in a glass beaker con-
taining 39 mL of PBS at 37.0  0.5 C, and were shaken at
100 rpm (shaker water bath, Selecta S.A., Spain). At pre-
fixed time intervals (15, 30, 45, 60, 90, 105, 120, 150, 180,
210, 240, 270, 300, 330, 360, 420, 480, 540, 600, 700, 800, 900,
1080 and 1800 min), 1 mL sample was withdrawn and ana-
lysed for insulin content by the previously described RP-
HPLC methodology. An equal volume of PBS (also kept at
37.0  0.5 C) was added after sampling to ensure sink
conditions.
Statistical analysis
Regarding the data gathered from insulin loading
experiments, the Levene test was used in the analysis of
the homogeneity of variance. With respect to the insulin
release investigations, the normal distribution of each
continuous variable was assessed by the ShapiroWilk
test. A factorial analysis of the variance was performed to
determine the effect on the dissolution efficiency of the
studied variables. Values with p 5 0.05 were considered
as significantly different.
Results and discussion
Particle geometry
All the insulin-loaded PLGA microparticles were of spher-
ical shape and has a smooth surface. Interestingly, the
formulation procedure did not seem to significantly
influence the final morphology of the microparticles
(Figure 2(a) and (b)). Mean particle size of the formulations
has been collected in Table 2. These formulations were in
the colloidal size range. Compared to FF, insulin-loaded
PLGA particles were significantly smaller upon formulation
by DE/SEV (F(1,12) 13.8; p 0.0034). On the contrary, the
FF technique allows obtaining insulin-loaded microparti-
cles with a clearly more narrow size distribution
(Figure 2(c)). In fact (Table 2), DE/SEV formulations were
characterized by absolute geometric standard deviation
(GSD) values 1.72 mm and, therefore, these formulations
could not be considered monodisperse. On the contrary,
GSD values of peptide-loaded microparticles prepared by
FF were  1.1 mm, thus confirming the great homogeneity
in size of FF formulations. Previous research reports have
associated GSD values 1.21.3 mm to monodisperse sam-
ples (Luque et al., 2009).
Electrokinetic properties
We begin this study analysing the effect of pH on the zeta
potential () of blank (non-loaded with insulin) PLGA
microparticles, and insulin-loaded PLGA microparticles
(prepared by DE/SEV, and FF), in the presence of 103 M
KNO3 (Figure 3(a) and (b)). Note that  values are negative
for the whole pH interval investigated and that they rise in
absolute value as the pH increases. This negative surface
charge has been previously associated to both the ioniza-
tion of weak acid groups of the polymer (presumably car-
boxylic end groups), and to dissociated end molecules of
the polysorbate surfactant Tween 80 used in the formula-
tion that remain adsorbed onto the PLGA surface even after
the cleaning procedure (Teixeira et al., 2005; Musumeci
et al., 2006; Okassa et al., 2007; Holgado et al., 2009). The
increasingly negative values and charge density may be
explainable by the effect of increasing OH ion concentra-
tion in the solution, which tends to favour a gain in protons.
In contrast, a decrease in absolute  as pH becomes more
acidic may be explainable by neutralization of the negative
regions as a result of chemical adsorption of increasing
numbers of H ions (Arias et al., 2007; Holgado et al., 2009).
With the aim of confirming these results, we also mea-
sured  as a function of KNO3 concentration at a constant
pH 6 (Figure 3(c) and (d)). The  values are negative for
the whole ionic strength interval investigated and they fall
in the absolute value as the KNO3 concentration increases,
due to the double-layer compression. The counterions
accumulate closer to the particle surface, such that the
double layer shrinks as concentration increases. This
leads to a lower electrical potential in the shear plane (or
slip surface) that limits the  value (Dillen et al., 2004; Arias
et al., 2009; Holgado et al., 2009).
More interestingly, no significant differences were found
between PLGA microparticles formulated by DE/SEV and
FF. Hence, it can be said that PLGA microparticles are,
from an electrokinetic point of view, indistinguishable
independently of the preparation technique. In addition,
 Insulin-loaded PLGA microparticles 435

Figure 2. Scanning electron microscopy photographs of insulin-loaded PLGA microparticles obtained by: (a) DE/SEV (formulation DE/SEV-4), and (b) FF
(formulation FF-12). (c) Histogram of particle size distribution of insulin-loaded PLGA micro particles obtained by DE/SEV and FF.

Table 2. Mean particle size (mm), insulin loading (%) and insulin entrapment efficiency (EE, %) of PLGA formulations prepared by DE/SEV or FF.
For personal use only.

Formulation Mean particle size (mm) CV (%) GSD (mm) Insulin loading (%) CV (%) EE (%) CV (%)

DE/SEV-1 1.03  0.84 61.46 1.65 0.53  0.01 1.89 53.87  1.54 2.86
DE/SEV-2 1.04  0.85 75.10 1.70 0.57  0.02 3.51 57.58  2.02 3.51
DE/SEV-3 0.49  0.31 80.66 1.99 2.85  0.03 1.05 59.52  0.52 0.89
DE/SEV-4 0.48  0.36 80.26 1.98 2.92  0.04 1.36 61.27  0.64 1.05
FF-1 1.32  0.14 10.21 1.11 3.32  0.06 1.81 69.82  1.23 1.76
FF-2 1.37  0.13 10.18 1.11 3.80  0.17 4.47 79.90  3.52 4.41
FF-3 1.26  0.11 8.40 1.08 3.53  0.08 2.26 74.30  1.69 2.28
FF-4 1.27  0.12 8.67 1.09 3.99  0.10 2.51 83.96  2.11 2.52
FF-5 1.31  0.11 9.31 1.10 3.86  0.06 1.55 81.16  1.35 1.66
FF-6 1.20  0.14 11.62 1.18 4.08  0.03 0.73 85.71  0.55 0.65
FF-7 1.21  0.15 12.56 1.13 4.12  0.03 0.72 86.62  0.53 0.61
FF-8 1.28  0.12 9.36 1.10 4.29  0.02 0.46 90.19  0.32 0.36
FF-9 1.20  0.14 11.50 1.18 4.54  0.04 0.88 95.37  0.75 0.79
FF-10 1.21  0.14 11.21 1.17 4.60  0.01 0.21 96.64  0.21 0.21
FF-11 1.32  0.17 12.47 1.13 4.68  0.02 0.43 98.39  0.32 0.33
FF-12 1.23  0.11 9.38 1.10 4.74  0.01 0.21 98.95  0.21 0.21

Note: CV (%), coefficient of variation and GSD (mm), geometric standard deviation.

our results clearly showed the great similarity between the
electrokinetics of non-loaded PLGA microparticles and all
the formulations of insulin-loaded PLGA microparticles.
This suggests that insulin was not adsorbed onto the
PLGA surface and, consequently, a very efficient insulin
entrapment into the microparticle core has occurred
(Nicoli et al., 2001; Dillen et al., 2004; Teixeira et al., 2005;
Dez and Tros de Ilarduya, 2006; Gomez-Gaete et al., 2007;
Holgado et al., 2009).
Surface thermodynamics
Table 3 collects experimental data from the analysis of
surface thermodynamics of the polymeric microparticles
by AFM. PLGA microparticles were found to be less hydro-
phobic than polystyrene (Das et al., 2010). Interestingly, the
hydrophobicity relative values of insulin-loaded PLGA
microparticles synthesized by FF were more similar to the
ones of polystyrene, compared to microparticles formu-
lated by DE/SEV. This indicates the higher hydrophobic
character of PLGA microparticles prepared by FF in
comparison to the ones obtained by DE/SEV. It could be
the consequence of a more significant incorporation
(adsorption) of hydrophilic PVA molecules onto PLGA sur-
face when the microparticles are prepared by DE/SEV
(Sahoo et al., 2002). This would be due to the
higher amount of PVA used in the formulation of PLGA
microparticles by DE/SEV.
 436 M. J. Cozar-Bernal et al.

Figure 3. Zeta potential of blank (non-loaded with insulin) PLGA microparticles, and insulin-loaded PLGA microparticles prepared by DE/SEV and FF as a
function of pH in the presence of 103 M KNO3 ((a) and (b), respectively), and as a function of the molar concentration of KNO3 at pH  6 ((c) and (d),
respectively). Formulations: blank (*), DE/SEV-1 (#), DE/SEV-2 (h), DE/SEV-3 (), DE/SEV-4 (), FF-1 (#), FF-2 (h), FF-3 (), FF-4 (), FF-5 (m), FF-6 (
),
FF-7 (n), FF-8 (5), FF-9 (^), FF-10 (S), FF-11 (o) and FF-12 (p).
For personal use only.

Table 3. Adhesion force (nN) and relative values of hydrophobicity (through Peyers patches) of particulate drug delivery sys-
of polystyrene and insulin-loaded PLGA microparticles formulated by
tems of 1 mm. Even more, the negative surface charge of
DE/SEV or FF.
the microparticles could facilitate its oral absorption
Adhesion force (nN) Relative values of through the intestinal membrane, thanks to a more intense
hydrophobicity electrostatic interaction between insulin-loaded PLGA
Polystyrene 50.2  0.1 0.33
microparticles and enterocytes. Finally, the hydrophobic
DE/SEV-1 129  0.2 0.85 character of the PLGA microcarriers is expected to further
DE/SEV-2 131  0.2 0.86 contribute to oral absorption of the peptide-loaded poly-
DE/SEV-3 133  0.2 0.88 meric particles. Preceding research reports have docu-
DE/SEV-4 132  0.2 0.87 mented a decreased gastrointestinal uptake in
FF-1 103.2  0.2 0.68
FF-2 102.4  0.2 0.67
hydrophilic microparticles. Hence, we could suggest that
FF-3 101.5  0.2 0.66 our polymeric microplatform could improve the oral
FF-4 103.5  0.2 0.68 absorption of insulin. However, in vitro and in vivo studies
FF-5 105.2  0.2 0.69 are needed to completely characterize cellular uptake of
FF-6 101.9  0.3 0.67
the microparticles and its biological fate.
FF-7 104.8  0.3 0.69
FF-8 105.3  0.2 0.69
FF-9 103.6  0.1 0.68
FF-10 104.9  0.2 0.69 Insulin loading to PLGA microparticles
FF-11 105.7  0.2 0.69
FF-12 104.3  0.2 0.68
Regarding PLGA microparticles formulated by DE/SEV,
Note: The adhesion force of probe-mica was determined to be 151.8 nN. two key parameters were studied: the polymer concentra-
tion, and the pH of the PVA solution (Table 2). Insulin
loading to PLGA was reduced as the amount of polymer
dissolved in the organic phase was increased. On the
Taking into account the information coming from the contrary, fixing an alkaline pH value in the PVA solution
complete physicochemical characterization done to the determined higher insulin entrapment efficiencies. The
insulin-loaded PLGA microparticles, we can hypothesize later effect was determined to be statistically significant
the biological fate of the formulations upon oral adminis- (F(1,8) 12.5; p 0.008), and it could be due to a stronger
tration (Hillery and Florence, 1996; Gaumet et al., 2010). electrostatic interaction between the positively charged
Concretely, particle size is not expected to be a limiting insulin macromolecules and the negatively charged
factor to oral absorption of the microcarrier, as previous PLGA surface. As a result, these alkaline pHs could
investigations have described gastrointestinal absorption minimize insulin leakage from polymer droplets

(Kawashima et al., 1999). Finally, no significant interaction
between both factors was observed (F(1,8) 1.6; p 0.242).
More importantly, greater insulin entrapment efficiency
was observed when PLGA microparticles were prepared by
FF (Table 2). For instance, the statistical comparison of
DE/SEV-3 and DE/SEV-4 with FF-1 and FF-2 formulations
demonstrated greater benefits coming from the prepara-
tion of the microparticles by FF with respect to peptide
entrapment efficiency (F(1,5) 14407.6818; p 5 0.0001).
Compared to DE/SEV, insulin-loaded PLGA microparticles
are obtained in one step by FF. Thus, the possibility of
protein loss during the synthesis procedure is minimized.
Similar conclusions have been previously reported when
lidocaine entrapment efficiencies were investigated in
PLGA particles prepared by these methodologies
(Holgado et al., 2008).
With respect to the effect of the formulation conditions
on insulin incorporation into PLGA microparticles, we
investigated the influence of the pH, and ionic strength of
the focusing fluid and the pH of the PVA solution in the
collected bath (Table 2). A statistically significant positive
influence was observed on peptide entrapment efficiency
of the enhancement of pH and ionic strength values of the
focusing fluid (F(2,26) 290.8; p 5 0.001; and, F(1,26) 47.1;
p 5 0.001, respectively), and of pH values of the PVA solu-
tion (F(1,26) 92.5; p 5 0.001). An alkaline focusing fluid
For personal use only.
would induce a reduction in the hydrophilic character of
the peptide (Leo et al., 1998, Kawashima et al., 1999, Pinto-
Reis et al., 2007). Similarly, high ionic strength values in the
focusing fluid will further decrease the hydrophilic charac-
ter of insulin, due to the loss of the hydrated shell around
the biomacromolecule and the reduction in its surface
charge (Fan et al., 2006). Under these conditions, the less
hydrophilic nature of the peptide will prevent its diffusion
out of the focusing fluid, thus improving its entrapment
into the PLGA microparticle.
The effect of the pH of PVA solutions on insulin entrap-
ment efficiency strongly depends on the composition of the
focusing fluid (F(2,26) 49.8; p 5 0.001) (Figure 4(a)).
Concretely, when the pH of the PVA solution was increased
from 6.5 to 9.0, insulin entrapment efficiency was
enhanced up to 13.7%, 4.8% and 0.9% when water, PBS
pH 7.4, and PBSpH 9.0 were used as focusing fluids,
respectively. Similarly, the effect of the composition of
the focusing fluid on insulin loading was significantly con-
ditioned by the pH of PVA solutions (Figure 4(b)).
Compared to water as a focusing fluid, when the pH of
the PVA solution was fixed to 9.0, an enhanced insulin
entrapment efficiency was observed upon using PBSpH
9.0 as focusing fluid, which was less important than when
PBSpH 7.4 was used (19.4% and 34.4%, respectively).
The enhancement in the insulin loading values was less
important when the pH of the focusing fluid and of the
PVA solution were closer. However, the highest insulin
entrapment efficiencies were obtained when both pHs
were adjusted to 9.0. Finally, no significant difference on
peptide entrapment efficiency was detected between the
pH and the ionic strength of the focusing fluid
(F(2,36) 0.736; p 0.608), and between the ionic strength
Insulin-loaded PLGA microparticles 437
of the focusing fluid and the pH of PVA solution
(F(2,36) 0.736; p 0.608). Thus, it can be stated that the
effect of the pH of the focusing fluid, and the pH of the PVA
solution on insulin loading efficiencies were not condi-
tioned by the ionic strength of the focusing fluid.
Stability of insulin loaded into PLGA microparticles
CD spectra of insulin prior to and after its loading into
PLGA microparticles is depicted in Figure 5. A minimum
222 nm in the spectra of insulin was observed, which is
typical of -helical proteins. The intensity and shape of the
CD spectra of pure insulin and insulin loaded to the micro-
carrier were almost indistinguishable. Negligible changes
could be associated to interferences coming from the poly-
mer. Hence, it can be said that the entrapment of the pep-
tide into PLGA did not alter the secondary (-helical)
structure of insulin, independent of the synthesis proce-
dure (Emami et al., 2009).
The tertiary structure of insulin was investigated by fluo-
rescence spectroscopy (Figure 6). No significant modifica-
tions were ascertained between the spectra of pure insulin
and insulin loaded to PLGA, independent of the synthesis
procedure. Experimental results indicated that tyrosine was
externally located in the polypeptide structure, which was
in agreement with the near-UV CD data (Gok and Olgaz,
2004; Sajeesh and Sharma, 2006, Nyambura et al., 2009,
Yong et al., 2009). Finally, less intense fluorescence bands
detected when insulin is loaded into PLGA by DE/SEV
could be attributed to the smaller entrapment efficiency
obtained following this synthesis procedure.
Release of insulin from PLGA microparticles
Release profiles of drug molecules and biomacromolecules
from PLGA particles have been reported to be severely con-
ditioned by PLGA characteristics (i.e. size, molecular
weight, surface thermodynamics, to cite just a few) and
by the mechanisms involved in the release process (e.g.
drug desorption, surface and/or bulk degradation, drug
diffusion, which could even simultaneously occur) (Luan
and Bodmeier, 2006; Sahana et al, 2008). Therefore, it is
very difficult to establish a general trend, and several
important controversies can be easily found between the
drug release profiles reported in the literature.
Independent of the synthesis procedure, insulin release
from PLGA microparticles follows a biphasic profile char-
acterized by an initial fast (burst) peptide release, followed
by a much slower release phase (Figure 7). Such insulin
release could be the consequence of diffusion-cum-degra-
dation mediated processes: the first phase was supposed to
be due to insulin diffusion through the external side of the
polymeric matrix (Ibrahim et al., 2005), and peptide release
during the second phase may result from polymer degra-
dation, from drug diffusion through the matrix, or both
(Mittal et al., 2007; Holgado et al., 2008). More interestingly,
faster release rates were observed in the formulations pre-
pared by DE/SEV: 75% of the peptide loaded was released
in less than 2 h, while the remaining 25% of insulin
 438 M. J. Cozar-Bernal et al.

Figure 4. Insulin entrapment efficiency into PLGA microparticles: (a) influence of the composition of focusing fluid (pH and ionic strength) on the effect of
the pH of the PVA solution and (b) influence of the pH of the PVA solution on the effect of the composition of the focusing fluid (pH and ionic strength).
For personal use only.

Figure 5. CD spectra of insulin, insulin degradation products, blank (non-loaded with insulin) PLGA microparticles and insulin-loaded PLGA formulations
DE/SEV-4 and FF-12.

Figure 6. Fluorescence spectra of insulin, and insulin-loaded PLGA formulations DE/SEV-4 and FF-12.

loaded was slowly liberated during the next 4 h.
On the contrary, insulin release from PLGA microparticles
synthesized by FF was complete after 10 h. These differ-
ences in the drug release profiles could be attributed to the
Figure 7. Release of insulin (%) from PLGA microparticles as a function of
the incubation time (min) in PBS (pH 7.4  0.1) at 37.0  0.5 C.
Formulations: DE/SEV-1 (#), DE/SEV-2 (), DE/SEV-3 (m), DE/SEV-4
(^), FF-1 (h), FF-2 (), FF-3 (
) and FF-4 (S). Lines are guides to the eye.
For personal use only.
Figure 8. Influence of the formulation conditions (polymer concentration and pH of PVA solution) on insulin dissolution efficiency.
Figure 9. Release of insulin (%) from PLGA microparticles as a function of the incubation time (min) in PBS (pH 7.4  0.1) at 37.0  0.5 C. Formulations:
FF-4 (#), FF-8 (h) and FF-12 (m). Lines are guides to the eye.
Insulin-loaded PLGA microparticles 439
characteristics of the FF formulations. In general, the
greater the hydrophobic character (the degradation rate
decreased as the hydrophobicity of a polymeric matrix is
higher), slightly bigger the particle size (responsible for a
smaller surface area/volume ratio in the polymer, which
leads to reduced buffer penetration into the matrix;
Mittal et al., 2007; Arias et al., 2008; Holgado et al., 2008;
Corrigan and Li, 2009).
The preparation conditions exclusively influenced insu-
lin release from PLGA formulated by DE/SEV. It was
observed that microparticles obtained by this method
exhibited a significant decrease in insulin release efficiency
when the polymer concentration (F(1,8) 13.49; p 5 0.006),
and the pH of PVA solution (F(1,8) 30.54; p 0.001) were
increased in their formulation conditions (Liu et al., 2006;
Hamishehkar et al., 2009). For instance, as the PLGA
concentration was increased from 0.5% to 1%, the dissolu-
tion efficiency decreased 7% and 5.9% when the pH of
PVA solution was 6.5 and 9.0, respectively (Figure 8).
However, the effect of the PLGA concentration on the
dissolution efficiency was independent of the influence of
the pH of PVA solution (F(1,8) 0.252; p 0.629).
 440 M. J. Cozar-Bernal et al.
Finally, we have appreciated different dissolution pro-
files when comparing the insulin-loaded PLGA formula-
tions prepared by FF. For clarity, we have only depicted
insulin release profiles from formulations FF-4, FF-8 and
FF-12, as we did not observe significant differences in the
insulin release behaviour when these microparticles were
prepared using different ionic strengths, and PVA solutions
with diverse pH values. Very interestingly, it was observed
that the greater the amount of insulin in the microparticles,
the lower was the release rate (Figure 9). In detail, although
insulin release was a biphasic process (as previously com-
mented), 55% insulin loaded to FF-4 was released within
2 h, while the remaining 45% was liberated during the
next 8 h; 40% was released within 2 h from FF-8, while
the remaining 60% was liberated during the next 16 h; and,
finally, 30% insulin loaded to FF-12 was released within
2 h, while the remaining 60% was liberated during the
next 28 h. This behaviour was also observed in DE/SEV
formulations.
Conclusions
We have defined the most significant differences between
insulin-loaded PLGA microparticles prepared by water-in-
For personal use only.
oil-in-water (w/o/w) DE/SEV method, and by FF tech-
nique. Compared to the former procedure, it has been
found that FF allowed the formulation of insulin-loaded
PLGA microparticles with a more narrow size distribution
and a greater hydrophobicity. In addition, PLGA micro-
particles prepared by FF were characterized by better insu-
lin vehiculization properties: higher insulin loading values
and slower insulin release profiles. We can consider that FF
technology will significantly improve the engineering of
insulin-loaded PLGA microparticles against diabetes.
Acknowledgements
Technical support from Ingeniatrics Tecnologas S.L.
(Spain) is gratefully acknowledged. Circular dichroism
analysis was feasible, thanks to Instituto de la Grasa
(CSIC, Seville, Spain). SEM and AFM analyses were possi-
ble, thanks to Centro de Investigacion, Tecnologa e
Innovacion of the University of Seville (CITIUS, Spain).
Declaration of interest
Financial support from Junta de Andaluca, Spain,
under projects P06-CTS-01688, and PE2008-FQM-3993 is
gratefully acknowledged.
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of this
article.
References
Arias JL, Gallardo V, Ruiz MA, Delgado AV. Ftorafur loading and controlled
release from poly(ethyl-2-cyanoacrylate) and poly(butylcyanoacrylate)
nanospheres. Int J Pharm, 2007;337:28290.
Arias JL, Gomez-Gallo A, Delgado AV, Gallardo V. Study of the stability of
Kollidon SR suspensions for pharmaceutical applications. Colloids Surf
A, 2009;338:10713.
Arias JL, Lopez-Viota M, Gallardo V, Ruiz MA. Chitosan nanoparticles as a
new delivery system for the chemotherapy agent tegafur. Drug Dev Ind
Pharm, 2010;36:74450.
Arias JL, Ruiz MA, Lopez-Viota M, Delgado AV. Poly(alkylcyanoacrylate)
colloidal particles as vehicles for antitumour drug delivery: A compara-
tive study. Colloids Surf B, 2008;62:6470.
Bernkop-Schnurch A. The use of inhibitory agents to overcome the enzy-
matic barrier to perorally administrated therapeutic peptides and pro-
teins. J Control Release, 1998;52:116.
Bhardwaj V, Hariharan S, Bala I, Lamprecht A, Kumar N, Panchagnula R,
Kumar N. Pharmaceutical aspects of polymeric nanoparticles for oral
delivery. J Biomed Nanotechnol, 2005;1:23558.
Bloemendal M, Jiskoot W. 2005. Circular dichroism spectroscopy.
In: Jiskoot W, Crommelin D, eds. Methods for structural analysis of pro-
tein pharmaceuticals. Arlington: AAPS Press, pp. 83130.
Corrigan OI, Li X. Quantifying drug release from PLGA nanoparticulates.
Eur J Pharm Sci, 2009;37:47785.
Cui F, Shi K, Zhang L, Tao A, Kawashima Y. Biodegradable nanoparticles
loaded with insulin-phospholipid complex for oral delivery: Preparation,
in vitro characterization and in vivo evaluation. J Control Release,
2006;114:24250.
Das T, Becker T, Nair BN. Measurements on hydrophobic and hydrophilic
surfaces using a porous gamma alumina nanoparticle aggregate
mounted on atomic force microscopy cantilevers. Thin Solid Films,
2010;518:276974.
Dez S, Tros de Ilarduya C. Versatility of biodegradable poly(D,L-lactic-co-
glycolic acid) microspheres for plasmid DNA delivery. Eur J Pharm
Biopharm, 2006;63:18897.
Dillen K, Vandervoort J, Van den Mooter G, Verheyden L, Ludwig A.
Factorial design, physicochemical characterisation and activity of cipro-
floxacin-PLGA nanoparticles. Int J Pharm, 2004;275:17187.
Emami J, Hamishehkar H, Najafabadi AR, Gilani K, Minaiyan M,
Mahdavi H, Nokhodchi A. A novel approach to prepare insulin-
loaded poly (lactic-co-glycolic acid) microcapsules and the protein sta-
bility study. J Pharm Sci, 2009;98:171231.
Fan YF, Wang YN, Fan YG, Ma JB. Preparation of insulin nanoparticles and
their encapsulation with biodegradable polyelectrolytes via the layer-by-
layer adsorption. Int J Pharm, 2006;324:15867.
Ganan-Calvo AM. Generation of steady liquid microthreads and
micron-sized monodisperse sprays in gas streams. Phys Rev Lett,
1998;80:2858.
Gaumet M, Gurny R, Delie F. Interaction of biodegradable nanoparticles
with intestinal cells: The effect of surface hydrophilicity. Int J Pharm,
2010;390:4552.
Gok E, Olgaz S. Binding of fluorescein isothiocyanate to insulin: A fluori-
metric labeling study. J Fluoresc, 2004;14:2036.
Gomez-Gaete C, Tsapis N, Besnard M, Bochot A, Fattal E. Encapsulation of
dexamethasone into biodegradable polymeric nanoparticles. Int J
Pharm, 2007;331:1539.
Hamishehkar H, Emami J, Najafabadi AR, Gilani K, Minaiyan M,
Mahdavi H, Nokhodchi A. The effect of formulation variables on the
characteristics of insulin-loaded poly(lactic-co-glycolic acid) micro-
spheres prepared by a single phase oil in oil solvent evaporation
method. Colloids Surf B, 2009;74:3409.
Hillery AM, Florence AT. The effect of adsorbed poloxamer 188 and 407
surfactants on the intestinal uptake of 60-nm polystyrene particles after
oral administration in the rat. Int J Pharm, 1996;132:12330.
Holgado MA, Arias JL, Cozar MJ, Alvarez-Fuentes J, Ganan-Calvo AM,
Fernandez-Arevalo M. Synthesis of lidocaine-loaded PLGA microparti-
cles by flow focusing. Effects on drug loading and release properties.
Int J Pharm, 2008;358:2735.
Holgado MA, Cozar-Bernal MJ, Salas S, Arias JL, Alvarez-Fuentes J,
Fernandez-Arevalo M. Protein-loaded PLGA microparticles engineered
by flow focusing: Physicochemical characterization and protein detec-
tion by reversed-phase HPLC. Int J Pharm, 2009;380:14754.
Ibrahim MA, Ismail A, Fetouh MI, Gopferich A. Stability of insulin during
the erosion of poly(lactic acid) and poly(lactic-co-glycolic acid) micro-
spheres. J Control Release, 2005;106:24152.
Johnson KL, Kendall K, Roberts AD. Surface energy and the contact of
elastic solids. Proc R Soc Lond A, 1971;324:30113.

Kawashima Y, Yamamoto H, Takeuchi H, Fujioka S, Hino T. Pulmonary
delivery of insulin with nebulized dl-lactide/glycolide copolymer (PLGA)
nanospheres to prolong hypoglycemic effect. J Control Release,
1999;62:27987.
Kumar N, Ravikumar MNV, Domb AJ. Biodegradable block copolymers.
Adv Drug Deliv Rev, 2001;53:2344.
Kumar PS, Saini TR, Chandrasekar D, Yellepeddi VK, Ramakrishna S,
Diwan P. Novel approach for delivery of insulin loaded poly(lactide-
co-glycolide) nanoparticles using a combination of stabilizers. Drug
Deliv, 2007;14:51723.
Leo E, Pecquet S, Rojas J, Couvreur P, Fattal E. Changing the pH of the
external aqueous phase may modulate protein entrapment and delivery
from poly(lactide-co-glycolide) microspheres prepared by a w/o/w sol-
vent evaporation method. J Microencapsul, 1998;15:42130.
Liu R, Huang SS, Wan YH, Ma GH, Su ZG. Preparation of insulin-loaded
PLA/PLGA microcapsules by a novel membrane emulsification method
and its release in vitro. Colloids Surf B, 2006;51:308.
Luan X, Bodmeier R. Influence of the poly(lactide-co-glycolide) type on the
leuprolide release from in situ forming microparticle systems. J Control
Release, 2006;110:26672.
Luque A, Perdigones F, Esteve J, Montserrat J, Ganan-Calvo AM, Quero JM. Sajeesh S, Sharma CP. Cyclodextrin-insulin complex encapsulated poly-
Reduction of dropet-size dispersion in parallel flow-focusing microde-
vices using a passive method. J Micromech Microeng, 2009;19:4502936.
Martn-Banderas L, Flores-Mosquera M, Riesco-Chueca P, Rodriguez-
Gil A, Cebolla A, Chavez S, Ganan-Calvo AM. Flow focusing: A versatile
technology to produce size-controlled and specific-morphology micro-
particles. Small, 2005;1:68892.
Martn-Banderas L, Rodriguez-Gil A, Cebolla A, Chavez S, Berdun- Shelma R, Paul W, Sharma CP. Development and characterization of self-
Alvarez T, Fernandez JM, Flores-Mosquera M, Ganan-Calvo AM.
Towards a high throughput production of uniform encoded microparti-
cles. Adv Mater, 2006;18:55964.
Matsuura J, Powers ME, Manning MC, Shefter E. Structure and stability of
insulin dissolved in 1-octanol. J Am Chem Soc, 1993;115:12614.
-glutamic acid) nanoparticles for oral insulin delivery.
Mittal G, Sahana DK, Bhardwaj V, Ravi Kumar MNV. Estradiol loaded
PLGA nanoparticles for oral administration: Effect of polymer molecular
For personal use only.
weight and copolymer composition on release behavior in vitro and
in vivo. J Control Release, 2007;119:7785.
Mundargi RC, Ramesh Babu V, Rangaswamy V, Patel P, Aminabhavi TM.
Nano/micro technologies for delivering macromolecular therapeutics
using poly(d,l-lactide-co-glycolide) and its derivatives. J Control
Release, 2008;125:193209.
Musumeci T, Ventura CA, Giannone I, Ruozi B, Montenegro L, Pignatello R,
Puglisi G. PLA/PLGA nanoparticles for sustained release of docetaxel.
Int J Pharm, 2006;325:1729.
Myers SR, Yakubu-Madus FE, Johnson WT, Baker JE, Cusick TS,
Williams VK, Tinsley FC, Kriauciunas A, Manetta J, Chen VJ. Acylation
of human insulin with palmitic acid extends the time action of human
insulin in diabetic dogs. Diabetes, 1997;46:63742.
Nicoli S, Santi P, Couvreur P, Couarraze G, Colombo P, Fattal E. Design of
triptorelin loaded nanospheres for transdermal iontophoretic adminis-
tration. Int J Pharm, 2001;214:315.
Norris DA, Puri N, Sinko PJ. The effect of physical barriers and properties
on the oral absorption of particulates. Adv Drug Deliv Rev,
1998;34:13554.
Insulin-loaded PLGA microparticles 441
Nyambura BK, Kellaway IW, Taylor KMG. Insulin nanoparticles: Stability
and aerosolization from pressurized metered dose inhalers. Int J Pharm,
2009;375:11422.
OBrien RW, White LR. Electrophoretic mobility of a spherical colloidal
particle. J Chem Soc Faraday Trans, 1978;2:160726.
Okassa LN, Marchais H, Douziech-Eyrolles L, Herve K, Cohen-Jonathan S,
Munnier E, Souce M, Linassier C, Dubois P, Chourpa I. Optimization of
iron oxide nanoparticles encapsulation within poly(D,L-lactide-co-gly-
colide) sub-micron particles. Eur J Pharm Biopharm, 2007;67:318.
Pinto-Reis C, Ribeiro AJ, Houng S, Veiga F, Neufeld RJ. Nanoparticulate
delivery system for insulin: Design, characterization and in vitro/in vivo
bioactivity. Eur J Pharm Sci, 2007;30:3927.
Sahana DK, Mittal G, Bhardwaj V, Ravikumar MNV. PLGA nanoparticles
for oral delivery of hydrophobic drugs: Influence of organic solvent on
nanoparticle formation and release behavior in vitro and in vivo using
estradiol as a model drug. J Pharm Sci, 2008;97:153042.
Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol
associated with poly (D,L-lactide-co-glycolide) nanoparticles affects
their physical properties and cellular uptake. J Control Release,
2002;82:10514.
methacrylic acid based nanoparticles for oral insulin delivery. Int J
Pharm, 2006;325:14754.
Santander-Ortega MJ, Bastos-Gonzalez D, Ortega-Vinuesa JL, Alonso MJ.
Insulin-loaded PLGA nanoparticles for oral administration: An in vitro
physico-chemical characterization. J Biomed Nanotechnol,
2009;5:4553.
aggregated nanoparticles from anacardoylated chitosan as a carrier for
insulin. Caborhydr Polym, 2010;80:28590.
Sonaje K, Chen YJ, Chen HL, Wey SP, Juang JH, Nguyen HN, Hsu CW,
Lin KJ, Sung HW. Enteric-coated capsules filled with freeze-dried chit-
osan/poly(
Biomaterials, 2010;31:338494.
Tatford OC, Gomme PT, Bertolini J. Analytical techniques for the evalua-
tion of liquid protein therapeutics. Biotechnol Appl Biochem,
2004;40:6781.
Teixeira M, Alonso MJ, Pinto MMM, Barbosa CM. Development and
characterization of PLGA nanospheres and nanocapsules containing
xanthone and 3-methoxyxanthone. Eur J Pharm Biopharm,
2005;59:491500.
Vadillo-Rodrguez V, Busscher HJ, Van der Mei HC, Vries J, Norde W. Role
of lactobacillus cell surface hydrophobicity as probed by AFM in adhe-
sion to surfaces at low and high ionic strength. Colloids Surf B,
2005;41:3341.
Yong Z, Yingjie D, Xueli W, Jinghua X, Zhengqiang L. Conformational and
bioactivity analysis of insulin: Freeze-drying TBA/water co-solvent
system in the presence of surfactant and sugar. Int J Pharm,
2009;371:7181.
Zhou XH, Po ALW. Peptide and protein drugs: I. Therapeutic
applications, absorption and parenteral administration. Int J Pharm,
1991a;75:97115.
Zhou XH, Po ALW. Peptide and protein drugs: II. Non-parenteral routes of
delivery. Int J Pharm, 1991b;75:11730.