Thyrotropin-releasing hormone loaded and chitosan engineered polymeric nanoparticles: Towards effective delivery of neuropeptides

Article abstract of DOI:10.1166/jnn.2016.12431

Thyrotropin-Releasing Hormone (TRH), a tripeptide amide with molecular formula L-pGlu-L-His-L-Pro-NH₂, is used in the treatment of brain/spinal injury and certain central nervous system (CNS) disorders, including schizophrenia, Alzheimer's disease, epilepsy, depression, shock and ischemia due to its profound effects on the CNS. However, TRH's therapeutic activity is severely hampered because of instability and hydrophilicity owing to its peptidic nature which results into ineffective penetration into the blood brain barrier. In the present study, we report the synthesis and stability studies of novel chitosan engineered TRH encapsulated poly(lactide-co-glycolide) (PLGA) based nanoformulation. The aim of such an encapsulation is to allow effective delivery of TRH in biological systems as the peptidase degrade naked TRH. The synthesis of TRH was carried out manually in solution phase followed by its encapsulation using PLGA to form polymeric nanoparticles (NPs) via nanoprecipitation technique. Different parameters such as type of organic phase, concentration of stabilizer, ratio of organic phase and aqueous phase, rate of addition of organic phase were optimized, tested and evaluated for particle size, encapsulation efficiency, and stability of NPs. The TRH-PLGA NPs were then surface modified with chitosan to achieve positive surface charge rendering them potential membrane penetrating agents. PLGA, PLGA-TRH, Chitosan-PLGA and Chitosan-PLGA-TRH NPs were characterized and analyzed using Dynamic Light Scattering (DLS), Transmissiom Electron Microscopy (TEM) and Infra-red spectroscopic techniques.

Full text of DOI:10.1166/jnn.2016.12431

Article
Journal of
Nanoscience and Nanotechnology
Vol. 16, 5324–5332, 2016
Copyright © 2016 American Scientific Publishers
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Printed in the United States of America
Thyrotropin-Releasing Hormone Loaded and
Chitosan Engineered Polymeric Nanoparticles:
Towards Effective Delivery of Neuropeptides
Sarabjit Kaur1, Avani Bhararia1, Krishna K. Sharma2, Sherry Mittal1, Rahul Jain2,
Nishima Wangoo3ꢀ∗, and Rohit K. Sharma1ꢀ∗
1Department of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University,
Sector-14, Chandigarh 160014, India
2Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Sector-67, S.A.S. Nagar, Mohali 160062, Punjab, India
3Department of Applied Sciences, University Institute of Engineering and Technology,
Panjab University, Sector-25, Chandigarh 160014, India
Thyrotropin-Releasing Hormone (TRH), a tripeptide amide with molecular formula L-pGlu-L-His-L-
Pro-NH2, is used in the treatment of brain/spinal injury and certain central nervous system (CNS)
disorders, including schizophrenia, Alzheimer’s disease, epilepsy, depression, shock and ischemia
Delivered by Ingenta to: Nanyang Technological University
due to its profound effects on the CNS. However, TRH’s therapeutic activity is severely hampered
IP: 193.0.129.26 On: Sun, 05 Jun 2016 13:23:59
because of instability and hydrophilicity owing to its peptidic nature which results into ineffective
Copyright: American Scientific Publishers
penetration into the blood brain barrier. In the present study, we report the synthesis and stability
studies of novel chitosan engineered TRH encapsulated poly(lactide-co-glycolide) (PLGA) based
nanoformulation. The aim of such an encapsulation is to allow effective delivery of TRH in biological
systems as the peptidase degrade naked TRH. The synthesis of TRH was carried out manually
in solution phase followed by its encapsulation using PLGA to form polymeric nanoparticles (NPs)
via nanoprecipitation technique. Different parameters such as type of organic phase, concentration
of stabilizer, ratio of organic phase and aqueous phase, rate of addition of organic phase were
optimized, tested and evaluated for particle size, encapsulation efficiency, and stability of NPs.
The TRH-PLGA NPs were then surface modified with chitosan to achieve positive surface charge
rendering them potential membrane penetrating agents. PLGA, PLGA-TRH, Chitosan-PLGA and
Chitosan-PLGA-TRH NPs were characterized and analyzed using Dynamic Light Scattering (DLS),
Transmissiom Electron Microscopy (TEM) and Infra-red spectroscopic techniques.
Keywords: Polymeric Nanoparticles, Thyrotropin-Releasing Hormone, Poly(lactide-co-glycolide)
PLGA, Chitosan, Neuropeptides, Nanoformulation.
1. INTRODUCTION
in the treatment of neurological diseases has become an
important area of research over the past two decades.3 This
includes the use of neuropeptides as primary pharmacolog-
ical agents in neurological disorders such as epilepsy, mak-
ing this approach an attractive proposition due to enhanced
potency and low toxicity of metabolites.4
Thyrotropin-releasing hormone (TRH), which is a sim-
ple neuropeptide with molecular formula L-pGlu-L-His-L-
Pro-NH2 (Fig. 1), is synthesized in the hypothalamus5 and
acts in the anterior pituitary to control levels of thyroid-
stimulating hormone (TSH, thyrotropin) and prolactin.
Neurological disorders are increasingly becoming one
of the major causes of diseases leading to fatal conse-
quences across the globe. As per a recent report by World
Health Organization (W.H.O.), neurological-based stroke
kills nearly 6.2 million people each year, out of which
more than 80% die in third world countries.1 It is esti-
mated that there are more than 50 million people that suf-
fer from epilepsy worldwide.2 The role of neuropeptides
∗Authors to whom correspondence should be addressed.
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1533-4880/2016/16/5324/009
doi:10.1166/jnn.2016.12431

Kaur et al.
TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
O
PLA for encapsulation of TRH has been recently utilized
with good results.31 The success of polymeric nanoparti-
cle formulation encouraged us to explore the encapsula-
tion of TRH using PLGA nanoparticles (PLGA NPs) and
to study the effect of such modification on the physico-
chemical nature of the resulting formulation. Hence, in this
paper we report a nanoparticle system that will also help
in improving the stability as the polymeric nanoparticles
(PLGA) protect the peptide drug from proteolytic cleav-
age by enzymes and thereby help in achieving low drug
dosage regime.
CONH2
NH
N
O
N
H
O
NH
N
Figure 1. Chemical structure of thyrotropin-releasing hormone (TRH).
Apart from the neuroendocrine action of TRH on the ante-
rior pituitary, TRH exerts profound effects on the central
nervous system (CNS).6 As a result of these effects, TRH
has been shown to alter neuronal excitability,7 enhance
transmitter release and turnover,8 increase CNS arousal,
increase blood pressure,9 body temperature10ꢀ11 and respi-
ration rate,12ꢀ13 alter body water and food intake, enhance
locomotor activity, and produce antinociception.14ꢀ15
Further, chitosan, a polysaccharide based on repeat-
ing units of randomly distributed D-glucosamine and
N -acetyl-D-glucosamine, has been employed for the sur-
face modification of PLGA NPs. The easy availability
of free amino groups in chitosan provides enough pos-
itive charge that allows it to bind negatively charged
surfaces/polymers and also undergo chelation with metal
ions. Moreover, the electrostatic adhesion to PLGA takes
effect as the chitosan surface is positively charged while
the PLGA with carboxyl groups has a negative surface
charge. This surface modification leads to reversal of sur-
face charge as negatively charged PLGA becomes pos-
itively charged on account of the free amino groups of
chitosan. In the present study, nano-precipitation technique
has been evaluated for the physical encapsulation of TRH
in PLGA NPs and further binding to chitosan (Fig. 2). Dif-
ferent variables involved in the nano-precipitation method
for synthesis of nanoparticles have been optimized, tested
and evaluated for particle size, encapsulation efficiency,
and stability. To best of our knowledge, no studies have
been conducted so far to examine or improve the physical
loading of TRH in PLGA/chitosan-PLGA NPs.
Most of the potential therapeutic applications of TRH
are based on its broad spectrum of stimulatory actions
within the CNS. Moreover, TRH has been found beneficial
in the treatment of Alzheimer’s disease,16 depression,17ꢀ18
epilepsy,19–21 spinal trauma22 etc. The use of TRH as a
CNS-active agent is, however, hampered by factors such
as its short half-life that does not allow its effective pen-
etration into blood brain barrier.23 Also TRH’s endocrine
effects are usually manifested at doses causing significant
cognitive improvement.24
To address these issues, there have been attempts to pro-
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Copyright: American Scientific Publishers
duce TRH-like compounds25ꢀ26 for separation of its CNS
and hormonal effects and to increase the drug availabil-
ity at the target site. However, the therapeutic efficacy of
TRH and similar compounds is compromised by instabil-
ity and hydrophilic nature owing to its peptidic nature. The
access of TRH to the CNS following oral or parenteral
administration is limited because of rapid degradation by
enzymatic action through proteolytic cleavage. These fac-
tors lead to administration of high dosage of peptide
for obtaining neuropharmacological effects often resulting
in adverse side effects due to the endocrine actions of
TRH.27
2. EXPERIMENTAL SECTION
2.1. Materials
PLGA (75:25) [mol wt (66,000–107,000)], Kolliphor P 188,
L-Prolinamide, L-Pyroglutamic acid, Boc-Histidine-OH,
N ,N -Diisopropylethylamine (DIPEA), N ,N ꢀ-Diisopropyl-
carbodiimide (DIC), N -hydroxy-5-norbornene-2,3-dicar-
boximide (HONB), Acetone, Acetonitrile, Tetrahydrofuran
(THF), Dichloromethane (DCM), and N ,N -Dimethyl-
formamide (DMF) were purchased from Sigma-Aldrich
(India). Ultrapure type I water of 18 ꢁm resistivity
used for experiments was obtained from Millipore Direct
Q3 system. All other chemicals used were of reagent
grade.
Over the recent times, there has been an increased inter-
est in nanosizing and particle manipulation of selected
drugs. In particular, polymeric nanoparticle formulation
has gained an increasing amount of attention in the fields
of drug delivery and pharmaceutics in recent years.28 There
has been an immense focus on the industrial utilization
of biodegradable polymer based nanoparticles as effective
drug delivery agents because of their ability to prolong
drug release, increase drug bioavailability, decrease drug
degradation and reduce drug toxicity.29 In this context, the
nanoparticle drug formulation research has focused on the
use of poly(lactic acid) (PLA), poly(D,L glycolide) (PLG),
and poly(lactide-co-glycolide) (PLGA) based nanoparti-
cles because of their tissue compatibility, low toxicity, and
high rate of hydrolysis.30 In the same context, the use of
2.2. Synthesis of Thyrotropin-Releasing
Hormone (TRH)
Boc-L-His-OH (1.2 mmol) (1) was dissolved in about
10–12 mL of dry DMF followed by addition of 1.8 mmol
(1.5 equiv.) of HONB and DIC each and the reac-
tion mixture was stirred for 15 minutes (Scheme 1).
This was followed by addition of 1.8 mmol (1.5 equiv.)
J. Nanosci. Nanotechnol. 16, 5324–5332, 2016
5325

TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
Kaur et al.
At
constant
rate
PLGA + TRH +
ACN
Chitosan
solution
Aqueous
stabilizer
solution
(Organic phase)
Stabilizer
Stirring
Reduced
pressure
Particle
hardening
(Aqueous phase)
Nanoprecipitation
Emulsion formation
Solvent evaporation
TRH-PLGA
Chitosan-TRH -PLGA
nanoparticles nanoparticles
Figure 2. Schematic diagram showing various stages involved in the preparation of chitosan-TRH-PLGA nanoparticles using nanoprecipitation
technique.
of L-Prolinamide and reaction mixture was stirred for
36 h at room temperature to afford Boc-L-His-L-Pro-NH2
(2), which was purified using column chromatography.
Further, the Boc group was deprotected using 10 mL
of 7N -methanolic HCl and the resulting substrate was
neutralized using 3 equiv. of DIPEA and then coupled with
L-Pyro-Glu-OH in the presence of HONB (1.5 equiv.) and
DIC (1.5 equiv.) under constant stirring for 36 hours at
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The final product 4 was purified using column chromatog-
Copyright: American Scientific Publishers
HN
raphy and all the synthesized compounds were analyzed
and characterized at each stage using H-NMR and mass
1
HN
N
N
spectrometry.
BocHN
N
a
BocHN
2.3. Synthesis of Polymeric Nanoparticles
To synthesize the polymeric nanoparticles, the nano-
precipitation technique with necessary modifications was
employed.32
O
H
O
O
NH2
Boc-L-His-OH
1
O
Boc-L-His-L-Pro-NH2
2
2.3.1. Synthesis of TRH-PLGA and PLGA NPs
For TRH-PLGA NPs, the polymer (PLGA) and drug
(TRH) were dissolved in a suitable organic solvent at a
concentration of 2.5 mg/mL and at various concentrations
(0.5–2 mg/mL), respectively to form the diffusing phase.
Further, 4 mL of this phase was then added to the dis-
persing phase (10 mL) at a specific rate by means of a
peristaltic pump positioned with the output tip directly in
the medium under moderate magnetic stirring. The dis-
persing phase was constituted from a liquid in which the
polymer is insoluble [here water—the non-solvent (NS)
containing a surfactant (Kolliphor P 188)] at various con-
centrations followed by stirring for 3–4 h, which allowed
complete evaporation of organic solvent. Finally, traces of
organic solvent were eliminated under reduced pressure at
b
HN
HN
N
N
H
O
N
N
H2N
.2HCl
c
H
O
N
N
O
O
NH2
NH2
O
L-pyro-Glu-L-His-L-Pro-NH2
O
L-His-L-Pro-NH2
3
(TRH)
4
ꢁ
40 C for 30 min. For PLGA NPs, TRH was omitted in
the first step with other steps followed as such in the above
procedure.
Scheme 1. Synthesis of TRH (a) DMF, DIC, HONB, Pro-NH2, 48 h,
rt (b) 7N-methanolic HCl (c) (i) DIPEA (3 equiv.) (ii) DIC (1.5 equiv.),
HONB (1.5 equiv.), p-Glu-OH (1.5 equiv.), DMF, 24–36 h, rt.
5326
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Kaur et al.
TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
2.3.2. Synthesis of TRH-PLGA-Chitosan NPs
2.7. Zeta Potential Determination
The Zeta potential of nanoparticles was determined by
using Zeta Sizer (Nano S-90, Malvern Instruments, UK).
The samples were placed in an electrophoretic cell, where
a potential of 150 mV was established and analyzed.
The samples were maintained at a constant temperature of
25ꢃ0 0ꢃ1 ꢁC, in all experiments.
A similar protocol, as reported in last section, was fol-
lowed for the preparation of chitosan coated PLGA
nanoperticles.33 Initially, chitosan solution (1 mg/mL) was
prepared by dissolving chitosan in 0.25 M HCl contain-
ing stabilizer at pH 5 as it was only sparingly soluble in
water at normal pH. Then, 4 mL of chitosan solution so
prepared was added to 10 mL of PLGA nanoparticles solu-
tion and the reaction mixture was incubated for 3–4 hours
to get chitosan coated PLGA (PLGA–CS) nanoparticles.
The freshly prepared nanoparticles were then centrifuged
four times for 15-min cycles at 6000 rpm and washed
with distilled water, in order to remove the unbound
chitosan.
2.8. Fourier Transform Infrared (FTIR) Spectroscopy
The FTIR transmission spectra of various samples
(chitosan, PLGA, TRH-PLGA-CS nanoparticles) were
obtained using a Thermo Scientific (Nicolet iS50 FT-
IR) spectrophotometer. TRH-PLGA-chitosan nanoparticles
were lyophilized to yield white powder, which was used
as such for FTIR analysis. PLGA, chitosan and TRH
were also analyzed in powder form. Each sample was
then scanned over wave number region of 400–4000 cm−1
and the characteristic peaks for different samples were
recorded.
2.4. Entrapment Efficiency and Drug Loading of
TRH-Loaded Nanoparticles
The amount of TRH present inside the nanoparticles was
estimated by using reverse phase high performance liquid
chromatography (RP-HPLC) (Waters 515HPLC model).
Briefly, 10 mL of prepared nanoparticles formulation was
centrifuged at 10,000 rpm. The supernatant collected was
analyzed using HPLC to determine amount of untrapped
TRH. 20 ꢂl of each sample was injected manually in the
injection port and was analyzed using a mobile phase of
acetonitrile-water-triflouroacetic acid mixture in the ratio
3. RESULTS AND DISCUSSION
3.1. Synthesis of Thyrotropin-Releasing
Hormone (TRH)
The solution phase based chemical synthesis of the TRH
was carried out in three steps. Initially, Boc-His-OH (1)
was coupled with L-prolinamide to obtain protected
15:85:0.1. TRH level was quantified by UV detection at
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dipeptide (2), which was subjected to Boc-deprotection
215 nm (photodiode array detecItPo:r)19w3it.h0.1fl2o9w.26raOteno: fSun, 05 Jun 2016 13:23:59
step leading to the formation of dipeptide (3) followed
Copyright: American Scientific Publishers
1 mL/min using HPLC pump (Waters 2996). The amount
by coupling with pyroglutamic acid to yield TRH (4)
of TRH in nanoparticles was determined from the peak
area correlated with the standard curve prepared under
identical conditions. The encapsulation efficiency (E.E.%)
was calculated by subtracting the amount of free drug in
the clear supernatant from the total amount of TRH added
to the nanosuspension.
(Scheme 1).
3.2. Synthesis of Polymeric Nanoparticles:
Investigation of Parameters Influencing
Nanoprecipitation
The polymeric nanoparticle preparation for effective
uptake by cells requires modulation of various parameters.
Size is one of the most important and effective parame-
ter in determining the cellular uptake and biological func-
tions of colloidal systems with general assumption that
a reduced size will usually result in significantly better
uptake of NPs by cells34 or better passage through mem-
brane. Moreover, it has been earlier demonstrated that
emulsion fabrication is a critical step during the prepara-
tion of NPs as the size of emulsion droplets is directly
relevant to final nanoparticle size.35ꢀ36 Various other factors
that may affect the size of polymeric NPs such as PLGA
NPs include the choice and concentration of stabilizer used
as well as the type of organic solvent used.37 Keeping these
facts in mind, synthetic and physicochemical studies were
carried out for generating a polymeric nanoparticle sys-
tem based on PLGA for the improved physical loading of
TRH thereby augmenting the stability of peptide drug and
achieving low drug dosage regime.
2.5. Particle Size Analysis
Mean particle size of the nanoparticles was determined
by photon correlation spectroscopy (PCS)/Dynamic light
scattering (DLS) using a Zeta Sizer (Nano S-90, Malvern
Instruments, UK). For size measurements, various formu-
lations of nanoparticles were further diluted with water to
50% and measured for a minimum of 120 s. All the sam-
plesꢁwere maintained at a constant temperature of 25ꢃ0
0ꢃ1 C, in all experiments and each batch was analyzed in
triplicates.
2.6. Transmission Electron Microscopy (TEM) Studies
Particle morphology was examined by transmission elec-
tron microscopy (TEM) using Hitachi (Model H-7500)
transmission electron microscope operating in accelerating
voltage of 120 kV. Samples (nanoparticulate suspensions)
were dropped onto carbon-coated copper grids, and after
complete drying, the samples were stained using 1% w/v
phosphotungstic acid.
For the synthesis of PLGA NPs, PLGA was dissolved
in various volatile organic solvents. The organic phase was
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TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
Kaur et al.
(A)
then added to the buffer solution with continuous stirring
at room temperature for 3–4 h to allow complete evap-
oration of the organic solvent followed by evaporation
at reduced pressure. Similarly, the preparation of TRH-
loaded nanoparticles was carried out in the same manner
by adding TRH along with PLGA. In order to achieve the
optimum particle size and high drug entrapment efficiency
during synthesis of polymeric NPs, effect of following
parameters was studied.
350
300
250
200
150
100
50
3.2.1. Effect of Organic Phase
To develop a successful formulation system comprising of
biodegradable NPs containing a pharmaceutical agent, the
choice of organic solvent becomes a critical step. While
choosing the suitable organic solvent, the parameters that
are most importantly considered include the ability of
organic solvent to dissolve polymer as well as drug and
its miscibility with water (for oil in water emulsion). Tox-
icity of the solvent chosen is also an important factor for
consideration as a trace amount of solvent is always left
in the final nanoparticle formulation prepared. Generally,
organic solvents that have low or no solubility in water
are considered to yield good quality spherical NPs with
non-porous and thus non-leaky surfaces. In addition, size
distribution of NPs as well as encapsulation efficiency is
closely affected by the ability of solvent to dissolve large
amount of polymer.38–40
0
acetone
acetonitrile
DCM
THF
Organic phase
(B)
600
500
400
300
200
100
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Thus, we envisioned a study in which different organic
Copyright: American Scientific0Publ1is:1h0ers
3:10
5:10
7:10
9:10
solvents (i.e., acetone, acetonitrile, tetrahydrofuran and
dichloromethane) were used as the organic phase to ana-
lyze the effect of solvent on particle size during NPs
formation. The bar graph shows the effect of different
solvents on particle size evaluated using dynamic light
scattering (DLS) (Fig. 3(A)). Although THF is consid-
ered relatively non-toxic among solvents under consider-
ation but the size of NPs obtained using THF was very
large as seen in the plot graph (Fig. 3(A)). On the other
hand acetonitrile,41 which also exhibits negligible toxicity
in trace amount, displayed minimum size of NPs in for-
mulation and hence it was chosen as a solvent of choice
for further studies.
ratio (organic phase:aqueous phase)
Figure 3. Bar graph showing the variation in size (diameter in nm) of
PLGA nanoparticles with change of organic solvent (A) and variation
in size (diameter in nm) of PLGA nanoparticles with change of ratio of
organic phase to aqueous phase used (B) as obtained using particle size
analyser.
of organic phase to aqueous phase (Fig. 3(B)). Therefore,
this concentration was chosen as the optimized condition
for further studies.
3.2.3. Effect of Rate of Addition of Organic Phase
During optimization studies, it was found out that even
the rate of addition of PLGA dissolved in organic phase
to aqueous phase had significant effect on the particle
size. Keeping this in mind, experiments were performed
in which variation in size of NPs with change of rate of
addition of organic phase was studied and the correspond-
ing results have been illustrated in a bar graph (Fig. 4(A)).
It is clearly evident from these results that at a lower rate
of addition of organic solvent phase to dispersion phase
(aqueous phase) leads to formation of smaller and uniform
particles in nano-precipitation process. The rate 0.5 ml/min
was found out to be appropriate for synthesis of small
sized and monodispersed NPs.
3.2.2. Effect of Ratio of Organic Phase to Aqueous
Phase on Particle Size
Ratio of organic solvent with respect to aqueous phase
plays an important role in nanoparticle formation. Using
acetonitrile as organic solvent, different organic to aqueous
phase ratios (1:10, 3:10, 5:10, 7:10, 9:10) were studied to
optimize particle size (Fig. 3(B)). The general observation
was that an increase in the organic phase to aqueous phase
ratio led to a slight decrease in mean diameter of nanopar-
ticles as the ratio was increased from 1:10 to 5:10. This
trend was followed till the ratio 5:10, beyond which the
particle size started to increase. Hence it was evident from
the plot that the particle size was least in case of 5:10 ratio
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Kaur et al.
TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
250
200
150
100
50
(A)
used for the size reduction of the droplets resulting in
bigger organic phase droplets, which consequently led to
increased particle size. Thus, it was found that the stabi-
lizer concentration is crucial in controlling the particle size
of synthesized NPs.
3.3. Nanoparticle Shape and Surface Morphology
Shape, size and surface morphology of unloaded PLGA
NPs as well as TRH-loaded PLGA NPs were examined
with the use of Transmission Electron Microscope (TEM)
(Fig. 5). Initially, the NPs were purified by repeated redis-
persion cycles and then centrifuged at 2000 rpm followed
by examination using TEM at 80 kV. The particle size was
found out to be approximately 60 nm for unloaded PLGA
NPs (Fig. 5(A)) and approximately 65 nm for TRH-loaded
PLGA NPs (Fig. 5(B)).
0
0.5
1.0
1.5
2.0
Rate of addition (ml/min)
(B)
500
In comparison to unloaded and TRH-loaded PLGA NPs,
chitosan coated NPs showed increase in size as the aver-
age diameter for these NPs was found out to be about
90 nm (Figs. 5(C and D)). This increase in size of NPs also
confirms the successful surface coating of chitosan layer
over PLGA NPs. The hydrodyanamic diameter obtained
using DLS of TRH-PLGA NPs and TRH-PLGA chitosan
NPs displayed the similar trend of increasing size. How-
ever, sizes of the NPs observed using DLS were found
to be much larger as compared to sizes observed in TEM
400
300
200
100
0
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images (Table I). This may be due to the large polymeric
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nature of NPs, which leads to much larger virtual hydrody-
C0o.p20y%right: American Scientific Publishers
0%
0.05%
0.10%
0.40%
namic circumference around the nanoparticles in solution
form.
Concentration of kolliphor (w/v)
Figure 4. Bar graph showing the variation in size (diameter in nm) of
PLGA nanoparticles with change of rate of addition of organic phase
(A) and variation in size (diameter in nm) of PLGA nanoparticles with
change of concentration of stabilizer (B) as obtained using particle size
analyser.
(A)
(B)
3.2.4. Effect of Stabilizer on Properties of
Synthesized Nanoparticles
The amount of stabilizer plays an important role in the
prevention of agglomeration of particles.42ꢀ43 NPs were
formulated with five different concentrations of buffer
solution (also acting as the stabilizer) i.e., 0%, 0.05%,
0.1%, 0.2% and 0.4% to study the effect of stabilizer in
properties of NPs. In the present study, an increase in
concentration of the stabilizer (Kolliphor P188) from 0 to
0.05% w/v made a significant decrease in the size of the
NPs prepared with the synthesized polymer (Fig. 4(B)).
The results exhibited the variation of particle size with
increasing concentration of stabilizer and it was observed
that 0.05% concentration of stabilizer resulted in small
and more stable NPs. As the concentration of stabilizer
is increased to higher values the particles size gradually
begins to increase. This might be due to the fact that at
higher concentrations, the increased viscosity of the aque-
ous phase might cause a hindrance to the energy input
100 nm
100 nm
(C)
(D)
100 nm
100 nm
Figure 5. TEM images of unloaded PLGA nanoparticles (A), TRH-
loaded PLGA nanoparticles (B), unloaded PLGA nanoparticles surface
coated with chitosan (C) and TRH loaded PLGA nanoparticles surface
coated with chitosan (D) prepared by nanoprecipitation technique.
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TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
Kaur et al.
Table I. Physicochemical characterization parameter values of TRH Loaded NPs.
Hydrodynamic diameter
(nm) (DLS)
Polydispersity
index (PDI)
Size (nm)
(TEM)
Zeta
potential (mV)
Entrapment
efficiency (EE) (%)
Drug loading
(DL) (ꢂg/mg)
PLGA NPs
TRH-PLGA NPs
TRH-PLGA-Chitosan NPs
128.9
133.3
269.1
0.178
0.103
0.227
60
65
90
−23ꢃ5
−10ꢃ3
+19ꢃ9
–
–
2.56
2.01
30.73
29.71
3.4. Physicochemical Characterization of PLGA,
TRH-PLGA and TRH-PLGA-Chitosan
3.6. Characterization of Synthesised
Nanoparticles Using FTIR
Nanoparticles
To confirm the existence of chitosan on PLGA NPs and
TRH in PLGA-chitosan nanoparticles, FTIR measure-
ments of the NPs were carried out (Fig. 6). A character-
istic band at 3285 cm−1 is attributed to –NH2 and –OH
groups stretching vibration in the chitosan matrix and the
band for amide at ∼1636 cm−1 is seen in the infrared
spectrum of native chitosan (Fig. 6(A)). Moreover, char-
acteristic peaks at ∼1454 cm−1 and ∼2883 cm−1 due to
amine functionality from chitosan was also found in case
of chitosan coated PLGA NPs suggesting a cationic modi-
fication of the PLGA NPs (Fig. 6(D)). The FTIR spectrum
of PLGA (Fig. 6(B)) shows strong peak at 1746 cm−1
which corresponds to the stretching vibrations of repeated
ester bonds, band at ∼2937 cm−1 due to C–H streching,
1456, 1380 cm−1 for –CH3, 1180, 1080, 882 (C–O stretch)
cm−1 while the band on 3100–3600 cm−1 belongs to the
OH group of the water.
The encapsulation efficiencies of TRH loaded PLGA NPs
and PLGA-CS NPs were determined by using High Perfor-
mance Liquid Chromatography (HPLC). Table I shows the
encapsulation efficiency at various types of nanoparticles
synthesized. All the encapsulation efficiency (EE) values
were calculated using formulas mentioned below and stan-
dard calibration curve was plotted using known quantities
of TRH employing area under the peak values obtained
from HPLC. The encapsulation efficiency (EE) and drug
loading (DL) were calculated as follows:
EE% = ꢄTotal amount of TRH used−Amount
of TRH in supernatant ꢄꢂgꢅꢅ
×ꢄTotal amount of TRH used (mg)ꢅ−1 ×100
Amount of TRH in nanoparticles ꢄꢂgꢅ
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Amount of PLGA (mg)
DL (w/w) =
The FTIR spectrum of TRH (Fig. 6(C)) shows broad
IP: 193.0.129.26 On: Sun, 05 Jun 2016 13:23:59
band between 3200–3600 cm−1 due to N–H stretching,
Copyright: American Scientific Publishers
peak at 2966, 2923 cm−1 due C–H stretching, a prevalent
peak at 2852 cm−1 due C O of amide bonds, peaks at
1733, 1679 cm−1 due to C O stretching, peaks between
1400 to 800 cm−1 due C–H bending and rocking.
The amount of TRH used in the nanoparticle preparation
was also first optimized to get maximum efficiency. It can
be noted that EE of 30.73% was observed in TRH-PLGA
loaded NPs when amount of TRH used was 2 mg per 6 mL
of total NPs prepared and the drug loading was found
out to be 2.56 ꢂg/mg of PLGA. Whereas in case of chi-
tosan coated PLGA NPs, the encapsulation efficiency was
29.71%, which is equivalent to TRH-PLGA NPs (Table I).
So it can safely interpreted that surface modification does
not hamper the encapsulation ability of NPs formulation.
Even drug loading in TRH-PLGA-Chitosan NPs is quite
similar to TRH-PLGA NPs (Table I).
By comparing the IR spectra for PLGA, TRH and TRH-
PLGA-chitosan NPs, it is confirmed that the synthesized
NPs are composed of PLGA and TRH. Besides, the pres-
ence of characteristic groups for chitosan confirms the
coating of chitosan on the TRH-PLGA NPs.
(D)
(C)
3.5. Surface Charge Analysis of
Synthesised Nanoparticles
The charge on the surface of PLGA, PLGA-TRH and
chitosan-TRH-PLGA NPs was analyzed by determining
their zeta potential values. The results indicated that PLGA
NPs as well as TRH-PLGA NPs demonstrated negative
zeta potential of −23.5 mV and −10.3 mV respectively,
while chitosan capped TRH-PLGA NPs exhibited positive
surface charge (+19.9 mV) (Table I). The amino groups
present on chitosan are responsible for the inversion of
charge. Thus, the zeta potential values confirm the asso-
ciation of positively charged chitosan to the surface of
negatively charged PLGA NPs.
(B)
(A)
3500
3000
2500
2000
1500
1000
500
Wavenumber(cm–1
)
Figure 6. FTIR spectra of chitosan (A), PLGA (B), TRH (C) and TRH-
PLGA-chitosan (D).
5330
J. Nanosci. Nanotechnol. 16, 5324–5332, 2016

Kaur et al.
TRH Loaded and Chitosan Engineered Polymeric Nanoparticles: Towards Effective Delivery of Neuropeptides
4. CONCLUSIONS
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Acknowledgments: This work was supported by the
Science Education and Research Board (SERB) of India
grant no. SB/SO/BB/0040/2013, University Grants Com-
mision (UGC), India grant no. 20-10 (12)/2012 (BSR)
and Department of Biotechnology (DBT), India, grant
no. BT/10460PFN/20/871/2013. Sarabjit Kaur and Sherry
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Delivered by Ingenta to: Nanyang Technological University
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Copyright: American Scientific Publishers
5332
J. Nanosci. Nanotechnol. 16, 5324–5332, 2016