Synthesis of a Nanoparticle System for the Enhanced Accumulation of Fluorescently-Labeled Amino Acids Encapsulated in Monodispersed Chitosan Nanoparticle System

Abstract

Poor in vivo bioavailability of nutrient is a major challenge in efficient delivery of nutraceutics. The increased bioavailability of nutraceuticals is prerequisite for efficient absorption by the gastrointestinal system. Nanotechnology-based approaches for nutraceutical applications could potentially increase absorption of nutrients and enhance its cellular accumulation due to its nanosize and promote better in vivo energy biodistribution. However, the dynamics of intracellular cell trafficking of nanoparticles and nutraceutical release has remain scarcely studied. This study describes a non-efficacious nanoparticle-mediated system for the encapsulation and delivery of fluorescently-labelled amino acids using tripolyphosphate as a crosslinker. Light scattering data showed successful formation of particle size as small as 65.69 nm with low polydispersity index (PDI) value of 0.178 at specific volume ratios of chitosan to tripolyphosphate. Following encapsulation, nanoparticle size and PDI value increased to 182.73 nm and 0.257 respectively discern successful accommodation of the fluorescently-labelled amino acid within its core. In vitro visualization of amino acids release and accumulation via fluorescence microscopy suggested that encapsulated amino acids were efficiently accumulated into Vero3 cell cytoplasm at 24 hours post treatment with localization in proximity to the cell nucleus. These results therefore suggested that the chitosan nanoparticle system developed was able to enhance the intracellular accumulation of glutamic acids and may serve as a suitable carrier for nutraceuticals delivery.

Full text

* To whom correspondence should be addressed.
Malays. Appl. Biol. (2017) 46(1): 171–175
SYNTHESIS OF A NANOPARTICLE SYSTEM FOR THE
ENHANCED ACCUMULATION OF FLUORESCENTLY-LABELLED
AMINO ACIDS ENCAPSULATED IN MONODISPERSED
CHITOSAN NANOPARTICLE SYSTEM
UMMU AFIQAH HASSAN1*, MOHD ZOBIR HUSSEIN2 and MAS JAFFRI MASARUDIN1
1Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
2Institute of Advanced Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor
*E-mail: afiqah_289@yahoo.com
Accepted 2 February 2017, Published online 31 March 2017
ABSTRACT
Poor in vivo bioavailability of nutrient is a major challenge in efficient delivery of nutraceutics. The increased bioavailability
of nutraceuticals is prerequisite for efficient absorption by the gastrointestinal system. Nanotechnology-based approaches for
nutraceutical applications could potentially increase absorption of nutrients and enhance its cellular accumulation due to its
nanosize and promote better in vivo energy biodistribution. However, the dynamics of intracellular cell trafficking of nanoparticles
and nutraceutical release has remain scarcely studied. This study describes a non-efficacious nanoparticle-mediated system for
the encapsulation and delivery of fluorescently-labelled amino acids using tripolyphosphate as a crosslinker. Light scattering
data showed successful formation of particle size as small as 65.69 nm with low polydispersity index (PDI) value of 0.178
at specific volume ratios of chitosan to tripolyphosphate. Following encapsulation, nanoparticle size and PDI value increased
to 182.73 nm and 0.257 respectively discern successful accommodation of the fluorescently-labelled amino acid within its
core. In vitro visualization of amino acids release and accumulation via fluorescence microscopy suggested that encapsulated
amino acids were efficiently accumulated into Vero3 cell cytoplasm at 24 hours post treatment with localization in proximity
to the cell nucleus. These results therefore suggested that the chitosan nanoparticle system developed was able to enhance the
intracellular accumulation of glutamic acids and may serve as a suitable carrier for nutraceuticals delivery.
Key words: nutraceuticals, chitosan nanoparticles, nanobiotechnology, amino acid, bioavailability
INTRODUCTION
Recently, there has been an exponential interest in
the development of nanotechnology-based delivery
carriers for anticancer drugs (Mazzaferro et al.,
2013), proteins and peptides (Gupta et al., 2013).
Effective oral delivery of these therapeutic
molecules is desirable but often challenging owing
to the many physical and physiological barrier
imposed primarily by the gastrointestinal track (GIT)
system leading to poor biological efficacy and
bioavailability (Bowman & Leong, 2006). This
limitation hence has brought to the rapid onset on
nanocarrier development towards improving their
therapeutic efficacy, where chitosan nanoparticles
have gained much attention owing to its special
physiochemical features. Owing to its special
physiochemical features such as low toxicity, high
biodegradability and biocompatibility, chitosan has
received a great traction in medical and biological
researches (Nadarajah et al., 2006). Chitosan is
reported to act as a permeation enhancer by
facilitating drug cellular uptake (Bowman & Leong,
2006) and its inherent mucoadhesive properties
promote drug absorption thus increase drug cellular
concentration and accumulation (Soane et al., 1999).
Over the past few years, bioactive compounds
derived from plant origins and herbs are extensively
used to treat common ailment and attributed as a
potent adjuvant for some chronic diseases (Gianfrilli
et al., 2012). In spite of their known potential to
provide added physiological and health benefits,
the low in vivo bioavailability of nutraceutics
limits their therapeutic applications (McClements
& Li, 2010). The increased bioavailability of
functional food ingredients such as amino acids is

172 AMINO ACIDS ENCAPSULATED IN MONODISPERSED CHITOSAN NANOPARTICLE SYSTEM
prerequisite for efficient absorption by GIT system.
Nanotechnology-based approaches for nutraceutical
applications could enhance the absorption of
nutrients by the digestive systems and increase
nutrients cellular accumulation as well. However,
the studies on mechanism of nutraceutics as well as
drug release and accumulation from nanocarriers are
remain limited. Release studies would provide better
elucidation on where and when nutraceutics are
released from nanocarriers. It is important to track
the release and localization of nutraceutics since
most therapeutic compounds need to be internalized
into cellular compartment and must achieve certain
concentration to exert their therapeutic effects. This
study proposes the synthesis of a chitosan-based
nanoparticle system and elucidation of this system
as a potential delivery carrier towards increasing in
vitro cellular accumulation and localization of
amino acids.
MATERIALS AND METHODS
Materials
Chitosan (low molecular weight), sodium
tripolyphosphate (TPP), L-glutamic acid (GA),
fluorescein 5(6)-isothiocyanate (FITC) and 4, 6-
diamidino-2, phenylindole dihydrochloride (DAPI)
in powder form were purchased from Sigma-Aldrich
(St Louis, MO, USA). RPMI media 1640, 0.25%
trypsin-EDTA and fetal bovine serum were
purchased from Gibco, Life Technologies (Grand
Island, USA). Sodium hydroxide pellet, 37%
hydrochloric acid and acetic acid glacial were
obtained from Friendmann Schmidt Chemicals
(Germany).
Synthesis of chitosan nanoparticles (CNPs)
CNPs were synthesized by ionic gelation routes
adapted and modified from Calvo et al (1997).
Chitosan (CS) was firstly prepared at concentration
of 1mg/mL. The CS was further diluted into 0.5
mg/mL final concentration an adjusted to pH 5.
Separately, TPP solution was prepared at
concentration of 1 mg/mL, and was then diluted
into 0.7 mg/mL. The resulting TPP solutions were
then adjusted to pH 2. CNPs were synthesized by
mixing 600 μL CS solution into increasing volume
of TPP solution (20 μL-300 μL). The resulting CNPs
were then purified by centrifugation at 13 000 rpm
for 20 minutes. The 40% of CNPs containing
supernatant were then collected and diluted with
60% deionized distilled water and was used for
further analysis and experimentation.
Synthesis of FITC-labelled GA (FITC-GA)
encapsulated CNPs
GA solution was prepared by dispersing 0.086
mg GA powder in 10 mL deionized distilled water
at 80ºC. The GA solution was subsequently diluted
into 0.05 M final concentration. FITC was used as
a labelling marker to tag GA. Briefly, 50 μL of FITC
was added to 200 μL GA and incubated for 10
minutes in the dark. Incorporation of FITC-GA into
CNP was achieved by adding the mixture of FITC-
GA into 600 μL CS solution (0.5 mg/mL) prior
addition of 250 μL of TPP (0.7 mg/mL). The mixture
was then thoroughly mixed to ensure a homogenous
suspension is obtained.
Analysis of CNP size and distribution by Dynamic
Light Scattering (DLS)
The size and distribution of synthesized CNPs
and FITC-GA encapsulated CNPs were analyzed by
Dynamic Light Scattering using Malvern Zetasizer
Nano S Instrument (Malvern Instruments, UK). The
CNPs liquid suspension was directly loaded into a
cuvette and the measurement of the particle size and
distribution was performed in triplicate for each
sample. Data are expressed as mean ± standard error
of mean (SEM). Any significant difference of particle
sizes between CNP samples prepared at different
TPP volume was analyzed by One Way Analysis of
Variance (ANOVA) whilst the significant difference
of particle size preceding and following
encapsulation was measured by paired t-test. The
p value < 0.05 is considered as significant.
In vitro visualization of GA release and
accumulation
FITC-GA encapsulated CNPs were prepared
following steps described earlier. Vero3 kidney
normal cells were seeded into 6 well plates at
density of 0.8 x105 cells/well and was incubated for
24 hours. On the next day, cells were treated with
200 μL of the samples and were incubated for 6
hours and 24 hours. Prior to the visualization, the
media from each well was discarded and the cells
were washed once with 1X PBS. Approximately,
2mL of 4% formaldehyde was added into each
well and was incubated for 3-5 minutes. The
formaldehyde was then directly discarded and the
cells were washed twice with 1X PBS. A volume
of 2mL of DAPI solution was added to each well.
The cells were then further incubated for 5 minutes.
Following incubation, the DAPI was discarded
and the cells were washed thrice with 1X PBS
before new media was added. The release and
accumulation of glutamic acids were observed under
fluorescence microscopy.

AMINO ACIDS ENCAPSULATED IN MONODISPERSED CHITOSAN NANOPARTICLE SYSTEM 173
RESULTS AND DISCUSSION
Particle size and distribution analyses by DLS
Formation of CNPs was mediated through
electrostatic interaction between protonated amine
groups of CS and anionic phosphate group of TPP
(Shu & Zhu, 2002). Figure 1 shows the particles size
produced at different TPP volume. The smallest CNP
sizes (65.69±4.86 nm) were produced at 250 μL
TPP addition to CS. The lowest PDI value of 0.178
(Figure 2) were also produced at this 250 μL TPP
volume. The significant differences of particle sizes
and PDI values were observed between CNPs
prepared at 20 μL and 200 μL TPP, 20 μL and 250
μL TPP, 250 μL and 300 μL TPP as shown in Figure
1 and Figure 2. PDI value reflects how mono-
dispersed the sample is and it is an indicator of
particle stability and distribution (Lim et al., 2013).
As shown in Figure 1 and Figure 2, the CNP size
and PDI value decreased as more TPP volume is
added to CS. The gradual decreases in particle sizes
were consistent with the increased availability of
anionic TPP site. Upon addition of TPP to CS,
protonated amine groups of CS start to crosslink
with TPP to form CNPs and the crosslinking density
is enhanced with further incorporation of TPP anion
(Liu & Gao, 2009). However, above 250 μL of TPP
addition, the particle size and PDI value increased
to 169.43±1.72 nm and 0.257, respectively. At this
point, only few free protonated amine groups of
CS are accessible by TPP anion (Ravikumara &
Madhusudhan, 2011). Further addition of TPP may
Fig. 2. Influence of TPP volume on PDI value.
Notes: The PDI value decreased with increasing TPP addition. The smallest PDI value was produced at
250 μL TPP volume. Error bars represent SEM from triplicate independent experiment.
aSignificant difference compared to 20 μL TPP addition. bSignificant difference compared to 50 μL TPP
addition. cSignificant difference compared to 250 μL TPP addition.
Fig. 1. Influence of TPP volume on particle size.
Notes: The particle size decreased with increasing TPP addition. The smallest particle size was produced
at 250 μL TPP volume. Error bars represent SEM from triplicate independent experiment.
aSignificant difference compared to 20 μL TPP addition. bSignificant difference compared to 50 μL TPP
addition. cSignificant difference compared to 250 μL TPP addition.

174 AMINO ACIDS ENCAPSULATED IN MONODISPERSED CHITOSAN NANOPARTICLE SYSTEM
disrupt the ionic linkages of already formed CNPs
which leads to formation of larger hydrodynamic
particle size (Fan et al., 2012), thus explaining the
increase in particle size and PDI value above 250
μL of TPP addition. These findings suggest that
CNPs with small size and low PDI value were
achieved at specific volume ratio of chitosan to TPP.
Figure 3 shows the size and PDI value of CNP
preceding and following encapsulation. The bar
graph and line graph represent CNP size and PDI
value respectively. The CNP size increased from
65.69±4.86 nm to 182.73±23.73 following
incorporation of FITC-GA into CNP. The increase
in particles size postulates successful loading of
FITC-GA within CNP. Encapsulation of FITC-GA
increased PDI value from 0.178 to 0.257. This
increase indicated the non-uniform distribution of
CNP. The CNP population constituted broad ranges
of particle size which consisted of free CNPs (< 100
nm) and FITC-GA encapsulated CNPs (> 100 nm).
In vitro accumulation of FITC-GA using
fluorescence microscopy
In order to test the potential of the synthesized
CNP system towards enhancing efficient
accumulation of GA into cells, the GA were tagged
with FITC. As shown in Figure 4A, fluorescence
signal was detected in cells treated with FITC-GA
Fig. 3. Particle size and PDI value of CNP before and following FITC-GA encapsulation.
Notes: The particle size and PDI value increased following encapsulation. Error bars represent SEM
from triplicate independent experiment.
*Significant difference of particle size of FITC-GA encapsulated CNP from CNP.
Fig. 4. In vitro cellular accumulation of FITC-GA (green) from CNPs into Vero3 cells.
Notes: Cells were treated with (A) FITC-GA encapsulated CNP for 24 hours and (B) free FITC- GA. DAPI was used
to stain the cell nucleus (blue). No green fluorescence signal was observed for cells treated with free FITC-GA.

AMINO ACIDS ENCAPSULATED IN MONODISPERSED CHITOSAN NANOPARTICLE SYSTEM 175
encapsulated CNPs. FITC-GA appears to accumulate
in cell cytoplasm and some of it resides in close
proximity to cell nucleus, while no fluorescence
signal was observed in cells treated with free
FITC-GA (Figure 4B). FITC-GA was found to only
accumulate in cells when incorporated within the
CNP carrier. No green fluorescence signal was
detected in cells treated with free FITC-GA
indicating that physical encapsulation within the
CNP is pivotal for efficient accumulation of GA into
cells. It was reported that encapsulation of molecules
into nanocarrier would enhance their internalization
into cells compared to free dosage form (Alexis et
al., 2008).
CONCLUSION
The formation of small, monodispersed CNP size
was achieved at specific volume ratio of CS to
TPP. The nanoparticle size increased following
encapsulation of FITC-GA indicates the successful
accommodation of the molecules within CNP core.
Visualization of FITC-labeled GA release and
accumulation from CNP system suggests the
potential of this CNP system towards enhancing
intracellular accumulation of GA thus may serve as
a potential carrier for food ingredients in
nutraceutical field as well as drug delivery of protein
and peptides for medical applications.
ACKNOWLEDGEMENTS
The author UAH would like to thank the Ministry
of Education, Malaysia for the myBrain15
scholarship, and Universiti Putra Malaysia for
provisions of a Graduate Research Fellowship. The
author MJM would like to acknowledge Universiti
Putra Malaysia for research funding under the
Inisiatif Putra Muda (IPM) scheme.
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