European Journal of Pharmaceutical Sciences 63 (2014) 29–35

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European Journal of Pharmaceutical Sciences

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

Surface modified PLGA nanoparticles for brain targeting of Bacoside-A

S. Jose a,⇑, S. Sowmya a, T.A. Cinu a,b, N.A. Aleykutty a, S. Thomas c, E.B. Souto d,e,f,⇑
a
University College of Pharmacy, Mahatma Gandhi University, Cheruvandoor Campus, Ettumanoor, Kottayam 686631, Kerala, India
b
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India
c
Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kottayam, India
d
Research Centre in Biomedicine (CEBIMED), Fernando Pessoa University, Praça 9 de Abril, 349, P-4249-004 Porto, Portugal
e
Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia, 296, P-4200-150 Porto, Portugal
f
Institute of Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Trás-os-Montes and Alto Douro, 5001-801 Vila-Real, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: The present paper focuses on the development and in vitro/in vivo characterization of nanoparticles com-
Received 13 April 2014 posed of poly-(D,L)-Lactide-co-Glycolide (PLGA) loading Bacoside-A, as a new approach for the brain
Received in revised form 3 June 2014 delivery of the neuroprotective drug for the treatment of neurodegenerative disorders (e.g. Alzheimer
Accepted 28 June 2014
Disease). Bacoside-A-loaded PLGA nanoparticles were prepared via o/w emulsion solvent evaporation
Available online 8 July 2014
technique. Surface of the nanoparticles were modified by coating with polysorbate 80 to facilitate the
crossing of the blood brain barrier (BBB), and the processing parameters (i.e. sonication time, the concen-
Keywords:
tration of polymer (PLGA) and surfactant (polysorbate 80), and drug-polymer ratio) were optimized with
Nanoparticles
Bacoside-A
the aim to achieve a high production yield. Brain targeting potential of the nanoparticles was evaluated
Poly(lactic-co-glycolic acid) by in vivo studies using Wistar albino rats. The nanoparticles produced by optimal formulation were
Blood brain barrier within the nanosized range (70–200 nm) with relatively low polydispersity index (0.391 ± 1.2). The
encapsulation efficiency of Bacoside-A in PLGA nanoparticles was 57.11 ± 7.11%, with a drug loading
capacity of 20.5 ± 1.98%. SEM images showed the spherical shape of the PLGA nanoparticles, whereas
their low crystallinity was demonstrated by X-ray studies, which also confirmed no chemical interactions
between the drug and polymer molecules. The in vitro release of Bacoside-A from the PLGA nanoparticles
followed a sustained release pattern with a maximum release of up to 83.04 ± 2.55% in 48 h. When com-
pared to pure drug solution (2.56 ± 1.23 lg/g tissue), in vivo study demonstrated higher brain concentra-
tion of Bacoside-A (23.94 ± 1.74 lg/g tissue) suggesting a significant role of surface coated nanoparticles
on brain targeting. The results indicate the potential of surface modified PLGA nanoparticles for the deliv-
ery of Bacoside-A to the brain.
Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction most important which is a triterpenoid saponin. Bacoside-A has

In the traditional Indian system of medicine ‘‘Ayurveda’’, Bacopa
monniera (Linn) has been used as a potent nerve tonic (Gupta et al.,
2014; Singh et al., 2014). It is thought to improve intelligence,
memory and functioning of sense organs, and it has also been used
to treat epilepsy, insomnia and asthma (Das et al., 2002; Deepak
and Amit, 2013; Thomas et al., 2013). The nootropic activity of this
plant extract has been attributed to the presence of two saponins,
namely Bacoside-A and Bacoside-B, of which the former is the
⇑ Corresponding authors. Tel.: +91 9447600750 (S. Jose). Address: Faculty of
Health Sciences of Fernando Pessoa University, Rua Carlos da Maia, 296, Office S.1,
P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637
(E.B. Souto).
E-mail addresses: sajanjose@hotmail.com (S. Jose), eliana@ufp.edu.pt
(E.B. Souto).
been reported to significantly improve acquisition, consolidation
and retention of memory (Singh and Dhawan, 1982). The ethanolic
extract of Bacopa has been found to increase the activity of antiox-
idative enzymes (e.g. superoxide dismutase (SOD), glutathione
peroxidase and catalase) in the frontal cortex, striatum and hippo-
campus of rats (Gubbannavar et al., 2013). Several studies have
been performed to prove its neuropharmacological effect (Pandey
et al., 2010). Its cognitive enhancing property makes it a potential
therapeutic for the treatment of neurodegenerative disorders
(Limpeanchob et al., 2008).
Bacoside-A has been tested for the treatment of neurodegener-
ative disorders, being Alzheimer disease one of the most common
neurodegenerative disorders causing dementia among the elderly
people. Oxidative stress and amyloid b are considered as the major
etiological and pathological factors in the initiation and promotion
of neurodegeneration in Alzheimer disease. However, the targeting

http://dx.doi.org/10.1016/j.ejps.2014.06.024
0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
30 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35
of therapeutics to the central nervous system (CNS) is limited by
restrictive mechanisms imposed at the blood brain barrier (BBB).
Opsonization by plasma proteins in the systemic circulation is also
an impediment to cerebral drug delivery (Liu et al., 2009; Roney
et al., 2005). The BBB, formed by the endothelial cells of the brain
capillaries coupled together by tight junctions, inhibits the trans-
port of 98% of all small drug molecules and 100% of large-molecule
pharmaceutics into the brain, and thus drug delivery to the brain is
restricted (Chen and Liu, 2012; Pardridge, 2003).
Nanoparticles proved to be a potential drug targeting system to
the brain with improved drug efficacy and reduced drug toxicity.
Due to their unique features, such as large surface to mass ratio,
their quantum properties and ability to carry and adsorb other
particles such as drug, proteins and probes, nanoparticles are
attractive for medical purposes. Among the various alternatives,
polymeric nanoparticles demonstrate to be promising candidates
as they are able of opening the tight junctions of the BBB, they
effectively disguise the membrane barrier limiting characteriza-
tions of the drug molecule thus prolonging drug release and pro-
tecting drugs against enzymatic degradation (Chen and Liu, 2012).
Biodegradable polymers are preferred as the matrix material for
nanoparticles, including e.g. poly(lactic-co-glycolic acid) (PLGA),
bovine serum albumin, and chitosan (Agyare et al., 2014). PLGA
is one of the most successfully used biodegradable polymers
because its hydrolysis leads to lactic acid and glycolic acid which
are endogenous and easily metabolized by the body via the Krebs
cycle. PLGA is approved by the US Food and Drug Administration
(FDA) and European Medicine Agency (EMA) in various drug deliv-
ery systems in humans (Danhier et al., 2012; Semete et al., 2010).
Polymeric nanoparticles in the size range of 100–200 nm are ideal
for brain targeting (Olivier, 2005; Wohlfart et al., 2012). The extent
of opsonization by organs with reticuloendothelial system (RES) is
reported to be low in this size range. Such low and narrow size
range also demonstrates high drug loading capacities and is able
to protect the incorporated drug against degradation, thus increas-
ing the chance of brain targeting and delivery (Modi et al., 2009;
Pardridge, 2002; Peng et al., 2013).
Surface modification of nanoparticles with certain surfactants,
such as polysorbates, has shown to enhance their brain targeting
potential (Kreuter et al., 1997; Wang et al., 2009). Polymeric nano-
particles coated with polysorbate 80 are reported to cross the BBB
by mimicking the low density lipoproteins (LDL), enabling them to
interact with the LDL receptor, resulting in the nanoparticles
uptake by brain endothelial cells (Gelperina et al., 2002; Nagpal
et al., 2013; Wohlfart et al., 2012).
2. Materials and methods
2.1. Materials
Bacoside-A was received as a gift sample from Sami Labs
Limited (Bangalore India). Poly (D,L-Lactide-co-Glycolide) [50:50]
was purchased from Purac Biomaterials (Gorinchem, The Nether-
lands) and was used without further purification. Polyvinyl alcohol
(PVA) was purchased from Research lab (Mumbai, India). Polysor-
bate 80 was procured from Nice Chemicals (Kochi, India). HPLC
grade Acetonitrile and Water were purchased from CDH chemicals
(New Delhi, India). All other chemicals and reagents used in this
study were of analytical grade.
2.2. Preparation of PLGA nanoparticles
PLGA nanoparticles loaded with Bacoside-A were prepared by a
modified version of the o/w emulsion solvent evaporation process
(Javadzadeh et al., 2010). PLGA polymer and Bacoside-A (drug
polymer ratio 1:1, 1:2, 1:3, 1:5) were dissolved in methanol-
dichloromethane mixture (1:2) which formed the organic phase.
The organic phase was emulsified with aqueous phase containing
PVA 2% (m/V) using an ultraprobe sonicator (Sonics vibra cell,
USA) at an output of 40 W for 5 min in an ice bath. To evaporate
the organic solvent, the formed nanosuspension was kept over-
night stirring in a magnetic stirrer. The resulting PLGA nanoparticle
suspension was ultra-centrifuged (Fourtech, Mumbai, India) at
13,000 rpm for 30 min at 4 °C and the sediment obtained was
washed with purified water and the washing process was repeated
thrice in order to remove the adsorbed/non-loaded drug molecules.
The washed nanoparticles were then freeze-dried using lyophilizer
(Subzero lab instruments, Chennai, India).
2.3. Coating of PLGA nanoparticles with polysorbate 80
Coating of PLGA nanoparticles (drug-polymer ratio 1:2) was
carried out by re-suspending nanoparticles in phosphate buffered
saline at a concentration of 20 mg/ml under constant stirring.
Relative to total suspension volume, polysorbate 80 was then
added to give a final solution of 1% (m/V) polysorbate 80, the
mixture was incubated for 30 min, and finally lyophilized. The
surfactant coating was confirmed by FTIR analysis (Anand et al.,
2010; Md et al., 2013; Mo et al., 2012).
2.4. Optimization of process parameters
2.4.1. Sonication time
The energy input is a fundamental step in order to obtain
emulsified systems. To study the influence of sonication time on
nanoparticles size distribution, sonication time was varied
between 2 and 7 min.
2.4.2. PLGA content
To verify the influence of the initial mass of polymer on the par-
ticles morphology, encapsulation efficiency (EE), and loading
capacity (LC), PLGA content was varied at 10, 20, 30 and 50 mg/ml.
2.4.3. Surfactant concentration
Polyvinyl alcohol (PVA) concentration was varied from 1% to 3%
(m/V) and the optimum concentration of PVA was evaluated to
reach a monodispersed emulsion, and therefore a with a narrow
particle size.
2.5. Determination of process yield
The process yield of nanoparticles was calculated by comparing
quantity of polymer and drug initially taken for the production
process, and the quantity of the nanoparticles finally obtained.
2.6. Determination of encapsulation efficiency and loading capacity
Encapsulation efficiency (EE) was calculated as the ratio of the
drug content in the freeze dried powder to the initial drug amount
added. The drug loading capacity (LC) was the ratio of the drug
content to the freeze dried powder (Mohammadi et al., 2010;
Narayanan et al., 2013). 5 mg of the freeze dried nanoparticles
was vortexed with 5 ml of methanol for 1 h and was filtered
through 0.22 lm membrane filter. Then, the drug content in the fil-
trate was analyzed by ultraviolet (UV) spectrophotometer at
278 nm (Mathew et al., 2010).
Real drug loading
Encapsulation Efficiency ðEEÞ ¼ Theoretical drug loading
 100
Loading Capacity ðLCÞ ¼ Weight of drug in nanoparticles
Weight of nanoparticles
 100
 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35
3. Characterization
3.1. Determination of particle size, zeta potential and morphology
The particle size and zeta potential of the prepared nanoparti-
cles (drug-polymer ratio 1:2) were analyzed by Zetasizer (Malvern,
UK). The freeze dried powder samples were suspended in water
and sonicated before measurement. The mean diameter and size
distribution of the resulted homogeneous suspension were
measured. Each value resulted from triplicate determinations.
Morphology of the nanoparticles was analyzed by scanning
electron microscopy (SEM) (JEOL JSM-6390, Tokyo, Japan). The
nanoparticles were fixed on adequate supports and coated with
platinum using platinum sputter module (JFC-1100, JEOL Ltd.), in
a higher vacuum evaporator for 5 min at 20 mA. Observations
under different magnifications were performed at 20 kv.
3.2. X-ray diffraction analysis
X-ray diffraction patterns were measured using X pert PRO,
PANalytical instrument, using Cu Ka rays with a voltage of 40 kV
and a current of 20 mA, over the 2h ranges 50–600, with a step
width 0.050 and a scan time of 2.0 s per step. X-ray diffraction pat-
tern were determined for the pure Bacoside-A, polymer (PLGA),
and for the optimized nanoparticles.
3.3. In vitro release profile of Bacoside-A from nanoparticles
The in vitro drug release from the surface modified nanoparticles
was performed applying the dialysis technique. 1 ml of nanoparticle
suspension (equivalent to 5 mg of Bacoside-A) was placed in a dial-
ysis bag (molecular weight cut off 10,000–12,000, Hi-Media, India)
sealed at both the ends and was soaked in 20 ml of phosphate buffer
solution (pH 7.4) and maintained at 37 °C ± 0.5 °C with continuous
stirring at 100 ± 5 rpm in a shaker. At predetermined time intervals,
aliquots were withdrawn from the release medium and replaced
with fresh phosphate buffer solution. The sample was assayed
spectrophotometrically for Bacoside-A at 278 nm. The studies were
performed in triplicate.
3.4. Animal studies
The targeting efficiency of polysorbate 80 coated Bacoside-A
loaded PLGA nanoparticles (drug: polymer ratio 1:2) were com-
pared with that of the free drug solution. Adult albino Wistar rats
were procured from the Animal House, University College of Phar-
macy, Cheruvandoor, M.G University, India after getting approval
from the Institutional Animal Ethical Committee Board, (IAEC no:
021/MPH/UCP/CVR/12). Rats weighing approximately 200–250 g
have been used for the in vivo study. The rats were maintained
on pellet diet and water ad libitum. Selected rats were kept on a
constant day and night cycle and fasted for 12 h before the study.
The animals were divided into 2 groups of 6 rats each and were
administered appropriate solution by intraperitoneal route.
The group-1 was given Bacoside-A pure drug and group-2 was
administered Bacoside-A bound with PLGA nanoparticles coated
with 1% (m/V) polysorbate 80. For the in vivo experiments, formu-
lations were resuspended in phosphate buffered saline. For the
surfactant coating, a solution of 1% (m/V) polysorbate 80 was
added, and the suspension was incubated for 30 min under stirring
prior to administration. All the formulations were given in a dose
equivalent to 20 mg/kg body weight. After 90 min of post injection,
the rats were sacrificed by cervical dislocation. The brain, liver,
spleen and kidneys were removed, weighed, and stored at
20 °C. The organs of each animal were homogenized separately
31
with 5 ml phosphate buffer containing methanol in the ratio
(10:1), respectively, and centrifuged. The drug content in the super-
natant was estimated using HPLC at 205 nm (Estella-Hermoso de
Mendoza et al., 2011; Srivastava et al., 2012; Wilson et al., 2008).
The results were expressed as mean ± standard deviation (SD). Sta-
tistical analysis was carried out by Mann–Whitney t-test. Analysis
was carried out using the trial version of Graph-PadÒ Prism v6
software. A difference was considered significant when P < 0.05.
4. Results and discussion
Bacoside-A-loaded PLGA nanoparticles were prepared by an o/
w emulsion solvent evaporation method. The sonication time,
PLGA and surfactant concentrations were optimized at 5 min,
20 mg/ml and 2%, respectively, based on the obtained particle size,
encapsulation efficiency (EE%), particle morphology and zeta
potential. When sonication time was increased from 2 to 7 min,
particle size decreased from 288.3 ± 2.23 nm to 173 ± 2 nm. Nano-
particles prepared with PLGA concentration of 20 mg/ml depicted a
spherical shape with minimal agglomeration and reaching an EE%
of 37.23 ± 1.45%. PVA taken at a concentration of 2% developed par-
ticles with a mean size of 207.7 ± 1.56 nm and zeta potential of
20.03 ± 0.95 mV.
Bacoside-A-loaded PLGA nanoparticles prepared at different
drug-polymer ratio showed an EE% ranging between
19.48 ± 2.08% and 76.94 ± 4.00%, with a drug LC% varying from
9.74 ± 1.03% to 20.5±.98% (Table 1). The highest drug loading of
20.5 ± 1.98% was obtained for formulation containing drug and
polymer in the ratio of 1:2, which was selected for further
in vitro and in vivo characterization studies. PLGA concentration
was fixed based on EE% and shape of the particles obtained by
SEM images. Table 1 shows the effect of PLGA concentration on
the encapsulation of the drug. The encapsulation of Bacoside-A
increased with an increase in PLGA concentration. The EE% was
found highest for F 7 (i.e. 50 mg/ml PLGA). The SEM images of
the formulations containing different concentrations of PLGA were
recorded. Nanoparticles prepared with 20 mg/ml of PLGA pre-
sented spherical shape without any agglomeration (Fig. 2 left).
However, nanoparticles prepared with 50 mg/ml of PLGA pre-
sented a non-spherical shape with presence of agglomerates
(Fig. 2, right) and the particle size was also found to increase.
Spherical particles with smooth surface have a relatively lower
surface to volume ratio, and thus a slower degradation rate when
compared to non-spherical particles. From these results, F5 con-
taining PLGA in the concentration of 20 mg/ml with an EE% of
37.23 ± 1.45 and spherical shaped particles with minimal agglom-
eration was selected over the other formulations. Formulations F6
and F7 showed higher EE% than F5 but exhibited a non-spherical
shape and agglomeration. Thus, PLGA concentration of 20 mg/ml
(F5) was selected.
PVA concentration was fixed based on the obtained particle size
and zeta potential. Formulations with different concentration of
PVA were prepared, and it was observed that with increase in
PVA concentration in the aqueous phase, the particle size
decreased from 288.4 ± 3.5 nm (1% PVA) to 91.5 ± 1.36 nm (3%
PVA). Table 2 shows that with the increase of PVA concentration,
the zeta potential increased from 31.06 ± 1.55 mV for 1% (m/V)
PVA to 6.65 ± 0.491 mV for 3% (m/V) PVA. The shift may be due
to the fact that increasing the PVA concentration, the number of
coating layers on the polymer surface will also increase, therefore
shielding the negative charge on the surface of the particles. From
the above results it was decided that 2% (w/v) PVA was best as a
surfactant.
The size of drug loaded nanoparticles (drug polymer ratio 1:2)
was 77 ± 1 nm with a zeta potential of 19 ± 0.89 mV (Fig. 1). From
32 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35

Table 1
Process yield, encapsulation efficiency, and loading capacity of Bacoside A in nanoparticles composed of different drug to polymer ratio (Formulation code F11 stands for (1:1)
drug–polymer ratio).

Formulation code Drug-polymer ratio Process yield (%w/w) Encapsulation efficiency (%w/w) Loading capacity (%w/w)
F1 1:1 15.00 19.48 ± 2.08 9.74 ± 1.03
F2 1:2 35.33 57.11 ± 7.11 20.50 ± 1.98
F3 1:3 47.00 61.73 ± 2.85 15.25 ± 0.98
F4 1:4 55.00 66.70 ± 2.40 13.33 ± 0.47
F5 1:5 42.00 76.94 ± 4.00 12.76 ± 0.68

n = 3, ±SD

Table 2 cles. SEM images have confirmed that the homogenous nature of
Comparison between PVA concentration and the zeta potential of nanoparticles particles, with a uniform distribution, without aggregation after
obtained with formulations F8, F9 and F10. lyophilisation. A spherical particle moving through a vessel does
Formulation code PVA % (w/v) Particle size (nm) Zeta potential (mV) not deviate from its streamline motion unless it experiences an
external force, which is not the case of non-spherical particles as
F8 1 288.4 ± 3.5 31.06 ± 1.55
F9 2 207.7 ± 1.56 20.03 ± 0.95 they are susceptible to segregation.
F10 3 91.5 ± 1.36 6.65 ± 0.49 The cumulative percentage release of Bacoside-A from PLGA
nanoparticles (drug-polymer 1:2) released up to 83.04 ± 2.55%
within 48 h in phosphate buffer pH 7.4 (Fig. 3). The release rate
of Bacoside-A loaded PLGA nanoparticles increased with increase
the results obtained in Table 1, the formulation with the highest of the drug-polymer ratio. Usually, the drug loaded carriers exhibit
drug LC% was selected for in vivo studies. The nanoparticles a biphasic release pattern.
(F12) showed an average particle size of 77.1 nm. Smaller particles The diffractogram of nanoparticles (drug polymer 1:2) con-
below 100 nm can easily cross BBB by preventing spleen filtration. firmed the drug loading into the formulated nanoparticles
Zeta potential which reveals the physical stability of the formula- (Fig. 4). The diffractogram of Bacoside-A exhibited sharp peaks,
tion was 19 ± 0.89 mV. Surface charge on the particles could indicating the crystalline nature of Bacoside-A, highlighting the
control the particles stability of the formulation through strong presence of drug loaded nanoparticles in an amorphous and disor-
electrostatic repulsion of particles, being therefore an important dered-crystalline status or in solid state solubilized form in the
factor to determine the in vivo interactions of nanoparticles with polymer matrix of nanoparticles.
the cell membrane. Positively charged particles have a higher The concentration of Bacoside-A in brain, liver, kidney and liver
tendency to attach and internalize compared to negatively or after intraperitoneal injection of polysorbate 80 coated nanoparti-
neutrally charged particles. cles and drug solution is shown in Fig. 5. The results indicate that
SEM analysis showed spherical shaped particles (Fig. 2). The only the polysorbate 80 coated nanoparticles were able to deliver
images have been obtained using a scanning electron microscope Bacoside-A in the brain considerably, with the concentration of
to determine the shape and surface morphology of the nanoparti- 23.94 ± 1.74 lg/g, 90 min after administration. The results also

Fig. 1. Particle size distribution (A, % intensity, average of 12 runs) and zeta potential (B, total counts, average of 3 runs) of the Bacoside-A loaded nanoparticles.
 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35 33

Fig. 2. SEM image of nanoparticles prepared with 20 mg/ml of PLGA (left, Formulation F5) and nanoparticles prepared with 50 mg/ml of PLGA (right, Formulation F7).

350
(A)

Counts (arbitrary units)

300

250

200

150

100

50

0
0 10 20 30 40 50 60 70
2 theta

300
(B)
Counts (arbitrary units)

250
Fig. 3. Cumulative percentage of Bacoside A released in phosphate buffer pH 7.4.
200

150
indicate that only 2.56 ± 1.23 lg/g of the drug was found in the
brain tissue of the group administered with drug solution. 100

The concentration of Bacoside-A, after the intraperitoneal

50
administration of pure drug solution, in the liver, spleen, and
kidneys were 13.16 ± 2.02, 3.93 ± 0.85 and 14.24 ± 3.8 lg/g, 0
0 10 20 30 40 50 60 70
respectively. However, when bound with polysorbate 80 coated
2 theta
PLGA nanoparticles, the concentration was 8.75 ± 2.77,
0.96 ± 0.132 and 7.66 ± 3.19 lg/g, respectively. Table 3 depicts
300
the average percentage of dose reached in each organ comparing (C)
the values obtained with polysorbate 80 coated Bacoside-A-loaded
Counts (arbitrary units)

250
nanoparticles (F12) and free Bacoside-A (S).
Solvent evaporation method has been selected to obtain PLGA 200
nanoparticles with homogenous size distribution. Nanoparticles
coated with polysorbate 80 are thought to cross the BBB via plasma 150

adsorption of apolipoproteins resulting in receptor mediated

100
endocytosis by brain capillary endothelial cells, as apolipoproteins
naturally cross the BBB. But another mechanism proposed is the 50
nanoparticle adherence to the cell membrane with subsequent
escape by the P-glycoprotein efflux system, and thus resulting in 0
0 10 20 30 40 50 60 70
endothelial cell uptake of nanoparticles (Benvegnu et al., 2012; 2-theta
Gelperina et al., 2002).
As the time of sonication increases, more energy is released Fig. 4. XRD pattern of pure Bacoside A (A) and Bacoside A loaded nanoparticles (B)
containing drug and polymer in the ratio 1:2.
during emulsification process, which leads to rapid dispersion of
polymeric organic phase as nanodroplets of small size. This may
be the reason for the decreased particle size observed with the the encapsulation (Song et al., 2008; Wang et al., 2013). But
increase of the sonication time. When sonication time of 7 min increase in PLGA concentration resulted in non-spherical particles
was set, metal contamination was observed in the nanosuspension with agglomeration, this may be due to the poor dispersion of
(Table 4). PLGA solution into the aqueous phase as the viscosity of dispersed
With an increase in PLGA concentration, the viscosity of the phase (polymer solution) increased. Coarse emulsions are obtained
organic phase increased, which caused the diffusion resistance at higher polymer concentrations, which lead to the production of
for drug molecules from organic phase to the aqueous phase, larger particles during the diffusion process. Thus, an optimum
thereby reducing the drug loss through diffusion and increasing concentration of 20 mg/ml of PLGA was selected.
34 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35

30 Table 4
formulation Optimization of sonication time.
Concentration of Bacoside A (µg/g)

standard
25 Formulation code Sonication time (min) Mean diameter (nm)
F1 2 288.3 ± 2.2
F2 5 207.7 ± 1.5
20
F3 7 173.0 ± 2.0

n = 3, ±SD
15

is in amorphous and disordered-crystalline phase or in solid state

10
5 particles exhibited a biphasic pattern an initial burst release and a
0 The release rate increased with increase in the drug-polymer ratio.
brain kidney liver
Groups
Fig. 5. Amount of drug per gram of brain, kidney, liver and spleen at 90 min after
i.p. administration (20 mg/kg) of Bacoside A loaded formulation and Pure Bacoside
A (S) (mean ± SD).
As the surfactant concentration increased, the zeta potential
moved towards higher positive charge, and positively charged
particles have a greater tendency to attach and internalize com-
pared to negatively charged particles; therefore, based on this fact
surfactant concentration was fixed at 2%.
A successful nanoparticle system should have a high loading
capacity to reduce the quantity of carrier required for administra-
tion. The drug loading capacity of nanoparticles was found to be
14.33 ± 4.00% against a theoretical drug loading of 29.97 ± 13.11%
depending on drug-polymer ratio.
As mentioned above, Bacoside-A loaded nanoparticles showed
an average particle size of 77 ± 1 nm. Smaller particles
(<200 nm), may facilitate the crossing of BBB by preventing spleen
filtration. The nanoparticles in this size range is ideal for brain tar-
geting as the extent of opsonisation by organs with RES is reported
to be low in this size range (Li et al., 2013; Olivier, 2005; Wohlfart
et al., 2012). Zeta potential was found to be in the range of
19 ± 0.89 mV. It reveals the physical stability of the formulation.
Positively charged particles have a higher tendency to attach and
internalize compared to negatively or neutrally charged particle.
Surface morphology studies have confirmed the nature of parti-
cles as homogenous, smooth and spherical in shape. A spherical
particle moving through a vessel does not deviate from its stream-
line motion unless it experiences an external force. But in case of
non-spherical particles they are susceptible to segregation.
X-ray diffraction is used to study interaction between drug and
polymer, as well as to study degree of sample crystallinity. The
diffractogram of bulk drug exhibited sharp peaks, indicating the
crystalline nature of Bacoside-A, whereas for PLGA shows the
amorphous nature. The diffractogram of drug loaded nanoparticles
solubilized form in the polymer matrix of nanoparticles.
The in vitro release profile of Bacoside-A from the loaded nano-
slower diffusion-initiated release due to concentration gradient.
spleen The initial burst release could mostly be caused by diffusion
release of drug particles from the surface of carriers. The release
rate and pattern of drug release from PLGA matrix is not only
dependent on diffusion of drug through the matrix but also on deg-
radation of PLGA. Therefore, drug loading, molecular weight, and
PLGA concentration are the major factors affecting the drug release
(Wischke and Schwendeman, 2008). All the formulations showed a
maximum of 30% burst release in the first 4 h. It is evident from the
drug release curves that drug-polymer ratio of 1:2 showed a max-
imum release of 83.04 ± 2.55% in 48 h. From the in vitro data a
biphasic release pattern has been obtained which is an initial burst
release followed by a sustained release which extends to 48 h.
Nearly 28% of the drug has been released within 2 h in in vitro,
which may be the initial burst release, but in case of in vivo studies
the release at 90 min shows a slower release rate which could be
due to the change in environmental conditions. It could also be
due to slower rate of particle degradation as PLGA is biodegradable
polymer. As a result, no empty particles would remain in the brain
as PLGA gets completely degraded to lactic acid and glycolic acid
which goes into metabolic pathways of the body. The in vitro
release study for all the 5 formulations with varying drug to poly-
mer ratio has been carried out. However, only the best formulation
was selected for in vivo study. The one which contains drug to
polymer in the ratio 1:2 was selected for in vivo, based on drug
loading.
PLGA nanoparticles coated with 1% polysorbate 80 were able to
deliver 10-fold more Bacoside-A into the brain when compared to
the free drug solution. This result is in strong support with the
findings of Kreuter et al., who reported that coating with polysor-
bate 80 resulted in increase in uptake of nanoparticles by a factor
of 5 (Borchard et al., 1994). The concentration of Bacoside-A of the
formulation group in kidney was lower than that of the drug
solution group. The reduced Bacoside-A concentration in kidney
of formulation group might decrease the renal toxicity, indicating
lower risk. Polysorbate 80 coated PLGA nanoparticles significantly
increased the uptake of Bacoside-A into the brain without any
accumulation in other organs in comparison to the drug solution.

Table 3
Average percentage of dose reached in each organ comparing the values obtained with P80 coated Bacoside A-loaded nanoparticles (F12) and free Bacoside A (S).

Sample Organ Peak area Conc. (lg/ml) Amount of drug in Amount per gram of Average percentage of dose
5 ml extract (lg) tissue (lg) reached in each organ (%)
P80 coated Bacoside A- Brain 137066.2 ± 19418.26 9.562978 ± 1.354794 47.81489 ± 6.183758 23.9467 ± 1.740055 0.9562
loaded Nanoparticles Kidney 28785.17 ± 11870.73 2.008314 ± 0.82821 10.04157 ± 4.141048 7.660576 ± 3.191621 0.20008
(F12) Liver 74013 ± 2488.12 5.163818 ± 1.736491 25.81909 ± 8.682453 8.753523 ± 2.773696 0.5163
Spleen 1346.333 ± 180.7735 0.093932 ± 0.012612 0.469662 ± 0.063062 0.9689 ± 0.132478 0.0093
Free Bacoside A (S) Brain 16223.67 ± 6190.637 1.13191 ± 0.473139 5.65955 ± 2.365695 2.562816 ± 1.231664 0.1131
Kidney 52578.83 ± 13408.79 3.668376 ± 0.935519 18.34188 ± 4.677593 14.24503 ± 3.801089 0.3668
Liver 97497.67 ± 12570.13 6.802321 ± 0.877006 34.0116 ± 4.385029 13.16161 ± 2.024464 0.6802
Spleen 5038.5 ± 1100.957 0.351531 ± 0.076813 1.757657 ± 0.384064 3.937453 ± 0.85978 0.0351
 S. Jose et al. / European Journal of Pharmaceutical Sciences 63 (2014) 29–35
5. Conclusions
Polysorbate 80 surface modified Bacoside-A loaded PLGA nano-
particles were successfully produced by solvent evaporation
method, showing an average particle size of 77 ± 1 nm and a zeta
potential value of 19 ± 0.89 mV. The drug release profile of the
optimized formulation depicted a maximum burs release of 30%
within the first 4 h with an extended profile up to 48% with a maxi-
mum release of 83.04 ± 2.55%. Encapsulation efficiency reached
57.11 ± 7.11%, with a drug loading capacity of 20.5 ± 1.98%. The
spherical-shapped PLGA nanoparticles coated with 1% polysorbate
80 were able to deliver 10-fold more Bacoside-A into the brain when
compared to the free drug solution. The high concentrations of
Bacoside-A achieved in the brain with the developed systems, trans-
late the potential of polysorbate 80 surface modified PLGA nanopar-
ticles for the treatment of diseases of the central nervous system.
6. Declaration of interest
Authors declare that there is no conflict of interest in this work.
Acknowledgements
The first author acknowledges the All India Council for Techni-
cal Education (AICTE), New Delhi, India, for the financial support
under Science Research Scheme (RPS 2012-13). The Portuguese
Science and Technology Foundation (FCT) and European Funds
(FEDER and COMPETE) are also acknowledged under the reference
PTDC/SAU-FAR/113100/2009.
References
Agyare, E.K., Jaruszewski, K.M., Curran, G.L., Rosenberg, J.T., Grant, S.C., Lowe, V.J.,
Ramakrishnan, S., Paravastu, A.K., Poduslo, J.F., Kandimalla, K.K., 2014.
Engineering theranostic nanovehicles capable of targeting cerebrovascular
amyloid deposits. J. Control. Release 185C, 121–129.
Anand, P., Nair, H.B., Sung, B., Kunnumakkara, A.B., Yadav, V.R., Tekmal, R.R.,
Aggarwal, B.B., 2010. Design of curcumin-loaded PLGA nanoparticles
formulation with enhanced cellular uptake, and increased bioactivity in vitro
and superior bioavailability in vivo. Biochem. Pharmacol. 79, 330–338.
Benvegnu, D.M., Barcelos, R.C., Boufleur, N., Pase, C.S., Reckziegel, P., Flores, F.C.,
Ourique, A.F., Nora, M.D., da Silva Cde, B., Beck, R.C., Burger, M.E., 2012.
Haloperidol-loaded polysorbate-coated polymeric nanocapsules decrease its
adverse motor side effects and oxidative stress markers in rats. Neurochem. Int.
61, 623–631.
Borchard, G., Audus, K.L., Shi, F., Kreuter, J., 1994. Uptake of surfactant-coated
poly(methyl methacrylate)-nanoparticles by bovine brain microvessel
endothelial cell monolayers. Int. J. Pharm. 110, 29–35.
Chen, Y., Liu, L., 2012. Modern methods for delivery of drugs across the blood–brain
barrier. Adv. Drug Deliv. Rev. 64, 640–665.
Danhier, F., Ansorena, E., Silva, J.M., Coco, R., Le Breton, A., Preat, V., 2012. PLGA-
based nanoparticles: an overview of biomedical applications. J. Control. Release
161, 505–522.
Das, A., Shanker, G., Nath, C., Pal, R., Singh, S., Singh, H., 2002. A comparative study in
rodents of standardized extracts of Bacopa monniera and Ginkgo biloba:
anticholinesterase and cognitive enhancing activities. Pharmacol. Biochem.
Behav. 73, 893–900.
Deepak, M., Amit, A., 2013. ‘Bacoside B’–the need remains for establishing identity.
Fitoterapia 87, 7–10.
Estella-Hermoso de Mendoza, A., Preat, V., Mollinedo, F., Blanco-Prieto, M.J., 2011. In
vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma.
J. Control. Release 156, 421–426.
Gelperina, S.E., Khalansky, A.S., Skidan, I.N., Smirnova, Z.S., Bobruskin, A.I., Severin,
S.E., Turowski, B., Zanella, F.E., Kreuter, J., 2002. Toxicological studies of
doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate)
nanoparticles in healthy rats and rats with intracranial glioblastoma. Toxicol.
Lett. 126, 131–141.
Gubbannavar, J.S., Chandola, H.M., Harisha, C.R., Khanpara, K., Shukla, V.J., 2013. A
comparative pharmacognostical and preliminary physico-chemical analysis of
stem and leaf of Bacopa monnieri (L.) Pennel and Bacopa floribunda (R.BR.).
Wettst Ayu 34, 95–102.
Gupta, P., Khatoon, S., Tandon, P.K., Rai, V., 2014. Effect of Cadmium on Growth,
Bacoside A, and Bacopaside I of Bacopa monnieri (L.), a Memory Enhancing
Herb. Sci. World J. 2014, 824586.
Javadzadeh, Y., Ahadi, F., Davaran, S., Mohammadi, G., Sabzevari, A., Adibkia, K.,
2010. Preparation and physicochemical characterization of naproxen–PLGA
nanoparticles. Colloids Surf., B 81, 498–502.
35
Kreuter, J., Petrov, V.E., Kharkevich, D.A., Alyautdin, R.N., 1997. Influence of the type
of surfactant on the analgesic effects induced by the peptide dalargin after its
delivery across the blood–brain barrier using surfactant-coated nanoparticles. J.
Control. Release 49, 81–87.
Li, J., Zhang, C., Fan, L., Jiang, X., Chen, J., Pang, Z., Zhang, Q., 2013. Brain delivery of
NAP with PEG-PLGA nanoparticles modified with phage display peptides.
Pharm. Res. 30, 1813–1823.
Limpeanchob, N., Jaipan, S., Rattanakaruna, S., Phrompittayarat, W., Ingkaninan, K.,
2008. Neuroprotective effect of Bacopa monnieri on beta-amyloid-induced cell
death in primary cortical culture. J. Ethnopharmacol. 120, 112–117.
Liu, G., Men, P., Kudo, W., Perry, G., Smith, M.A., 2009. Nanoparticle–chelator
conjugates as inhibitors of amyloid-b aggregation and neurotoxicity: a novel
therapeutic approach for Alzheimer disease. Neurosci. Lett. 455, 187–190.
Mathew, J., Soman, S., Sadanandan, J., Paulose, C.S., 2010. Decreased GABA receptor
in the striatum and spatial recognition memory deficit in epileptic rats: effect of
Bacopa monnieri and bacoside-A. J. Ethnopharmacol. 130, 255–261.
Md, S., Ali, M., Baboota, S., Sahni, J.K., Bhatnagar, A., Ali, J., 2013. Preparation,
characterization, in vivo biodistribution and pharmacokinetic studies of
donepezil-loaded PLGA nanoparticles for brain targeting. Drug Dev. Ind.
Pharm.
Mo, L., Hou, L., Guo, D., Xiao, X., Mao, P., Yang, X., 2012. Preparation and
characterization of teniposide PLGA nanoparticles and their uptake in human
glioblastoma U87MG cells. Int. J. Pharm. 436, 815–824.
Modi, G., Pillay, V., Choonara, Y.E., Ndesendo, V.M., du Toit, L.C., Naidoo, D., 2009.
Nanotechnological applications for the treatment of neurodegenerative
disorders. Prog. Neurobiol. 88, 272–285.
Mohammadi, G., Valizadeh, H., Barzegar-Jalali, M., Lotfipour, F., Adibkia, K., Milani,
M., Azhdarzadeh, M., Kiafar, F., Nokhodchi, A., 2010. Development of
azithromycin-PLGA nanoparticles: physicochemical characterization and
antibacterial effect against Salmonella typhi. Colloids Surf. B Biointerfaces 80,
34–39.
Nagpal, K., Singh, S.K., Mishra, D.N., 2013. Nanoparticle mediated brain targeted
delivery of gallic acid: in vivo behavioral and biochemical studies for protection
against scopolamine-induced amnesia. Drug Deliv. 20, 112–119.
Narayanan, D., Anitha, A., Jayakumar, R., Chennazhi, K.P., 2013. In vitro and in vivo
evaluation of osteoporosis therapeutic peptide PTH 1–34 loaded PEGylated
chitosan nanoparticles. Mol. Pharm.
Olivier, J.C., 2005. Drug transport to brain with targeted nanoparticles. NeuroRx 2,
108–119.
Pandey, R., Gupta, S., Tandon, S., Wolkenhauer, O., Vera, J., Gupta, S.K., 2010.
Baccoside A suppresses epileptic-like seizure/convulsion in Caenorhabditis
elegans. Seizure 19, 439–442.
Pardridge, W.M., 2002. Drug and gene targeting to the brain with molecular Trojan
horses. Nat. Rev. Drug Discov. 1, 131–139.
Pardridge, W.M., 2003. Blood–brain barrier drug targeting: the future of brain drug
development. Mol. Interv. 3 (90–105), 151.
Peng, Q., Zhang, S., Yang, Q., Zhang, T., Wei, X.-Q., Jiang, L., Zhang, C.-L., Chen, Q.-M.,
Zhang, Z.-R., Lin, Y.-F., 2013. Preformed albumin corona, a protective coating for
nanoparticles based drug delivery system. Biomaterials 34, 8521–8530.
Roney, C., Kulkarni, P., Arora, V., Antich, P., Bonte, F., Wu, A., Mallikarjuana, N.N.,
Manohar, S., Liang, H.-F., Kulkarni, A.R., Sung, H.-W., Sairam, M., Aminabhavi,
T.M., 2005. Targeted nanoparticles for drug delivery through the blood–brain
barrier for Alzheimer’s disease. J. Control. Release 108, 193–214.
Semete, B., Booysen, L., Lemmer, Y., Kalombo, L., Katata, L., Verschoor, J., Swai, H.S.,
2010. In vivo evaluation of the biodistribution and safety of PLGA nanoparticles
as drug delivery systems. Nanomed. Nanotechnol. Biol. Med. 6, 662–671.
Singh, H.K., Dhawan, B.N., 1982. Effect of Bacopa monniera Linn. (brahmi) extract on
avoidance responses in rat. J. Ethnopharmacol. 5, 205–214.
Singh, R., Ramakrishna, R., Bhateria, M., Bhatta, R.S., 2014. In Vitro Evaluation of
Bacopa monniera Extract and individual constituents on human recombinant
monoamine oxidase enzymes. Phytother. Res..
Song, X., Zhao, Y., Hou, S., Xu, F., Zhao, R., He, J., Cai, Z., Li, Y., Chen, Q., 2008. Dual
agents loaded PLGA nanoparticles: systematic study of particle size and drug
entrapment efficiency. Eur. J. Pharm. Biopharm. 69, 445–453.
Srivastava, P., Raut, H.N., Puntambekar, H.M., Desai, A.C., 2012. Stability studies of
crude plant material of Bacopa monnieri and quantitative determination of
bacopaside I and bacoside A by HPLC. Phytochem. Anal. 23, 502–507.
Thomas, R.B., Joy, S., Ajayan, M.S., Paulose, C.S., 2013. Neuroprotective potential of
Bacopa monnieri and Bacoside A Against dopamine receptor dysfunction in the
cerebral cortex of neonatal hypoglycaemic rats. Cell. Mol. Neurobiol..
Wang, C.X., Huang, L.S., Hou, L.B., Jiang, L., Yan, Z.T., Wang, Y.L., Chen, Z.L., 2009.
Antitumor effects of polysorbate-80 coated gemcitabine polybutylcyanoacrylate
nanoparticles in vitro and its pharmacodynamics in vivo on C6 glioma cells of a
brain tumor model. Brain Res. 1261, 91–99.
Wang, H., Jia, Y., Hu, W., Jiang, H., Zhang, J., Zhang, L., 2013. Effect of preparation
conditions on the size and encapsulation properties of mPEG-PLGA
nanoparticles simultaneously loaded with vincristine sulfate and curcumin.
Pharm. Dev. Technol. 18, 694–700.
Wilson, B., Samanta, M.K., Santhi, K., Kumar, K.P.S., Paramakrishnan, N., Suresh,
B., 2008. Targeted delivery of tacrine into the brain with polysorbate 80-
coated poly(n-butylcyanoacrylate) nanoparticles. Eur. J. Pharm. Biopharm. 70,
75–84.
Wischke, C., Schwendeman, S.P., 2008. Principles of encapsulating hydrophobic
drugs in PLA/PLGA microparticles. Int. J. Pharm. 364, 298–327.
Wohlfart, S., Gelperina, S., Kreuter, J., 2012. Transport of drugs across the blood–
brain barrier by nanoparticles. J. Control. Release 161, 264–273.