ArticlePDF Available

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

Authors:

Abstract and Figures

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.
Content may be subject to copyright.
* 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.
REFERENCES
Alexis, F., Pridgen, E., Molnar, L.K. & Farokhzad,
O.C. 2008. Factors affecting the clearance and
biodistribution of polymeric nanoparticles.
Molecular Pharmaceutics, 5(4): 505-515.
Bowman, K. & Leong, K.W. 2006. Chitosan
nanoparticles for oral drug and gene delivery.
International Journal of Nanomedicine, 1(2):
117.
Calvo, P., RemunanLopez, C., VilaJato, J.L. &
Alonso, M.J. 1997. Novel hydrophilic chitosan-
polyethylene oxide nanoparticles as protein
carriers. Journal of Applied Polymer Science,
63(1): 125-132.
Fan, W., Yan, W., Xu, Z. & Ni, H. 2012. Formation
mechanism of monodisperse, low molecular
weight chitosan nanoparticles by ionic gelation
technique. Colloids and Surfaces B: Bio-
interfaces, 90: 21-27.
Gianfrilli, D., Lauretta, R., Di Dato, C., Graziadio,
C., Pozza, C., De Larichaudy, J., Giannetta, E.,
Isidori, A.M. & Lenzi, A. 2012. Propionyl-L-
carnitine, Larginine and niacin in sexual
medicine: a nutraceutical approach to erectile
dysfunction. Andrologia, 44(s1): 600-604.
Gupta, S., Jain, A., Chakraborty, M., Sahni, J.K., Ali,
J. & Dang, S. 2013. Oral delivery of therapeutic
proteins and peptides: a review on recent
developments. Drug Delivery, 20(6): 237-246.
Lim, J., Yeap, S.P., Che, H.X. & Low, S.C. 2013.
Characterization of magnetic nanoparticle by
dynamic light scattering. Nanoscale Research
Letters, 8(1): 1-14.
Liu, H. & Gao, C. 2009. Preparation and properties
of ionically crosslinked chitosan nanoparticles.
Polymers for Advanced Technologies, 20(7):
613-619.
Mazzaferro, S., Bouchemal, K. & Ponchel, G. 2013.
Oral delivery of anticancer drugs III: formulation
using drug delivery systems. Drug Discovery
Today, 18(1): 99-104.
McClements, D.J. & Li, Y. 2010. Review of in
vitro digestion models for rapid screening of
emulsion-based systems. Food & Function, 1(1),
32-35.
Nadarajah, K., Lau, B.Y.C., Othman, O., Hasidah,
M.S. & Wan-Mohtar, W.Y. (2006). Charac-
terization of chitin deacetylase from fungus
Absidia butleri dr. Malaysian Applied Biology,
35(2): 59.
Ravikumara, N.R. & Madhusudhan, B. 2011.
Chitosan nanoparticles for tamoxifen delivery
and cytotoxicity to MCF-7 and Vero cells. Pure
and Applied Chemistry, 83(11): 2027-2040.
Shu, X.Z. & Zhu, K.J. 2002. The influence of
multivalent phosphate structure on the
properties of ionically cross-linked chitosan
films for controlled drug release. European
Journal of Pharmaceutics and Biopharma-
ceutics, 54(2): 235-243.
Soane, R.J., Frier, M., Perkins, A.C., Jones, N.S.,
Davis, S.S. & Illum, L. 1999. Evaluation of
the clearance characteristics of bioadhesive
systems in humans. International Journal of
Pharmaceutics, 178(1): 55-65.
... The formation of CNP occurred through ionic crosslinking between protonated amine groups of CS and anionic phosphate groups of the cross linker, Sodium Tripolyphosphate (TPP) [27,28]. When cross linking occurs, the protonated amine groups will associate with the anionic phosphate groups of TPP to form nanoparticles where the CS polymer will start to form spherical, nano-scaled particles. ...
Article
Full-text available
Conventional delivery of anticancer drugs is less effective due to pharmacological drawbacks such as lack of aqueous solubility and poor cellular accumulation. This study reports the increased drug loading, therapeutic delivery, and cellular accumulation of silibinin (SLB), a poorly water-soluble phenolic compound using a hydrophobically-modified chitosan nanoparticle (pCNP) system. In this study, chitosan nanoparticles were hydrophobically-modified to confer a palmitoyl group as confirmed by 2,4,6-Trinitrobenzenesulfonic acid (TNBS) assay. Physicochemical features of the nanoparticles were studied using the TNBS assay, and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analyses. The FTIR profile and electron microscopy correlated the successful formation of pCNP and pCNP-SLB as nano-sized particles, while Dynamic Light Scattering (DLS) and Field Emission-Scanning Electron Microscopy (FESEM) results exhibited an expansion in size between pCNP and pCNP-SLB to accommodate the drug within its particle core. To evaluate the cytotoxicity of the nanoparticles, a Methylthiazolyldiphenyl-tetrazolium bromide (MTT) cytotoxicity assay was subsequently performed using the A549 lung cancer cell line. Cytotoxicity assays exhibited an enhanced efficacy of SLB when delivered by CNP and pCNP. Interestingly, controlled release delivery of SLB was achieved using the pCNP-SLB system, conferring higher cytotoxic effects and lower IC50 values in 72-h treatments compared to CNP-SLB, which was attributed to the hydrophobic modification of the CNP system.
Article
Full-text available
Lung cancer has been recognized as one of the most often diagnosed and perhaps most lethal cancer diseases worldwide. Conventional chemotherapy for lung cancer-related diseases has bumped into various limitations and challenges, including non-targeted drug delivery, short drug retention period, low therapeutic efficacy, and multidrug resistance (MDR). Chitosan (CS), a natural polymer derived from deacetylation of chitin, and comprised of arbitrarily distributed β-(1-4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit) that exhibits magnificent characteristics, including being mucoadhesive, biodegradable, and biocompatible, has emerged as an essential element for the development of a nano-particulate delivery vehicle. Additionally, the flexibility of CS structure due to the free protonable amino groups in the CS backbone has made it easy for the modification and functionalization of CS to be developed into a nanoparticle system with high adaptability in lung cancer treatment. In this review, the current state of chitosan nanoparticle (CNP) systems, including the advantages, challenges, and opportunities, will be discussed, followed by drug release mechanisms and mathematical kinetic models. Subsequently, various modification routes of CNP for improved and enhanced therapeutic efficacy, as well as other restrictions of conventional drug administration for lung cancer treatment, are covered.
Article
The ionic gelation method was used to study the effect of the crosslinking agent, sodium tripolyphosphate on average particle size (Dp) and zeta potential (ζp) of chitosan microparticles (CSMP) unloaded and loaded with trans-cinnamaldehyde (TCIN). The obtained values of Dp and ζp trend as 117.6 ± 0.4 ≤ Dp ≤ 478.5 ± 3.5 nm and +27.8 ± 1.3 ≤ ζp ≤ +103.5 ± 4.2 mV, respectively. The entrapment efficiency of TCIN in CSMP was 9.1 ± 2.0% and 71.5 ± 2.9% was released after 360 min (pH = 6.5) which reveals a potential anti-cancer activity in acidic environment. Cytotoxicity of TCIN in DMSO (0–50 μM) was evaluated on MDCK and HeLa cell lines and exhibited low effect at either 24 or 48 h of exposure; whereas TCIN-loaded CSMP (0–50 μM) showed, after 24 h of exposure, 67.6 ± 7.0 and 64.5 ± 3.9% cytotoxicity for MDCK and HeLa cell lines, respectively. At 48 h of exposure, TCIN-loaded CSMP achieved 81.1 ± 0.26 and 77.9 ± 4.2% cytotoxicity for MDCK and HeLa cell lines, respectively.
Article
Full-text available
The chitin deacetylase enzyme that catalyses the conversion of chitin to chitosan via the deacetylation of N-acetylglucosamine residues has been found in a few Zygomycetes and insects. Currently, chitosan is produced from chitin through harsh thermochemical procedure that has their disadvantages. The development of a controllable process using the enzymatic deacetylation of chitinous materials presents an alternative process that enables, in principle, the preparation of novel chitosan polymers and oligomers. Here we report the partial purification of chitin deacetylase (CDA; EC 3.5.1.41) from Absidia butleri dr, a local fungal isolate. The partially pure enzyme obtained will be used to further elucidate the properties of CDA. Homogenization of the mycelia to obtain the cell-free crude extract of CDA by grinding with acid washed-sand produced a lower yield (17.6 mU/mL) compared to the use of a homogenizer (21.6 mU/mL). However, the former was chosen in this study to obtain the crude CDA as the method gave less contaminating proteins. Through a combination of anion exchange and gel filtration chromatography, the crude CDA was isolated as two isoenzymes, designated CDA1 and CDA2. The isoenzymes have apparent native molecular masses of 97.6 kDa (CDA1) and 221.7 kDa (CDA2). The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that CDA1 and CDA2 have yet to be purified to electrophoretic homogeneity. A modified method to determine the enzyme activity has also been described.
Article
Full-text available
Here we provide a complete review on the use of dynamic light scattering (DLS) to study the size distribution and colloidal stability of magnetic nanoparticles (MNPs). The mathematical analysis involved in obtaining size information from the correlation function and the calculation of Z-average are introduced. Contributions from various variables, such as surface coating, size differences, and concentration of particles, are elaborated within the context of measurement data. Comparison with other sizing techniques, such as transmission electron microscopy and dark-field microscopy, revealed both the advantages and disadvantages of DLS in measuring the size of magnetic nanoparticles. The self-assembly process of MNP with anisotropic structure can also be monitored effectively by DLS.
Article
Full-text available
There is increasing interest in understanding and controlling the digestion of emulsified lipids within the food and pharmaceutical industries. Emulsion-based delivery systems are being developed to encapsulate, protect, and release non-polar lipids, vitamins, nutraceuticals, and drugs. These delivery systems are also being used to control the stability and digestion of lipids within the human gastrointestinal tract so as to create foods that enhance satiety and reduce hunger. In vitro digestion models are therefore needed to test the efficacy of different approaches of controlling lipid digestion under conditions that simulate the human gastrointestinal tract. This article reviews the current status of in vitro digestion models for simulating lipid digestion, with special emphasis on the pH stat method. The pH stat method is particularly useful for the rapid screening of food emulsions and emulsion-based delivery systems with different compositions and structures. Successful candidates can then be tested with more rigorous in vitro digestion models, or using animal or human feeding studies.
Article
In this study, tamoxifen citrate-loaded chitosan nanoparticles (tamoxcL-ChtNPs) and tamoxifen citrate-free chitosan nanoparticles (tamoxcF-ChtNPs) were prepared by an ionic gelation (IG) method. The physicochemical properties of the nanoparticles were analyzed for particle size, zeta (ζ) potential, and other characteristics using photon correlation spectroscopy (PCS), zeta phase analysis light scattering (PALS), scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and differential scanning calorimetry (DSC). The variation in particle size was assessed by changing the concentration of chitosan, pentasodium tripolyphosphate (TPP), and the pH of the solution. The optimized tamoxcL-ChtNPs showed mean diameter of 187 nm, polydispersity of 0.125, and ζ-potential of +19.1 mV. The encapsulation efficiency (EE) of tamoxifen citrate (tamoxc) increased at higher concentrations, and release of tamoxc from the chitosan matrix displayed controlled biphasic behavior. Those tamoxcL-ChtNPs tested for chemosensitivity showed dose- and time-dependent antiproliferative activity of tamoxc. Further, tamoxcL-ChtNPs were found to be hemocompatible with human red blood cells (RBCs) and safe by in vitro cytotoxicity tests, suggesting that they offer promise as drug delivery systems in therapy.
Article
Abstract Advent of recombinant technology in protein synthesis has given birth to a new range of biopharmaceuticals. These therapeutic peptides and proteins are now emerging as an imperative part of various treatment protocols especially in the cancer therapeutics. Despite extensive research efforts, oral delivery of therapeutic peptide or protein is still a challenge for pharmaceutical industries and researchers. Number of factors including high proteolytic activity and low pH conditions of gastrointestinal tract act as major barriers in the successful delivery of intact protein/peptide to the targeted site. Low permeability of protein/peptide across the intestinal barrier is also a factor adding to the low bioavailability. Therefore, because of the short circulatory half-life exhibited by peptides in vivo, they need to be administered frequently resulting in increased cost of treatment and low patient compliance. Nano-carrier-based delivery presents an appropriate choice of drug carriers owing to their property to protect proteins from degradation by the low pH conditions in stomach or by the proteolytic enzymes in the gastrointestinal tract. This review focuses on recent aspects and patents on oral delivery of therapeutic proteins and peptides with special emphasis on nano-carrier-based approach.
Article
Chitosan nanoparticles were fabricated by a method of tripolyphosphate (TPP) cross-linking. The influence of fabrication conditions on the physical properties and drug loading and release properties was investigated by transmission electron microscopy (TEM), dynamic light scattering (DLS), and UV–vis spectroscopy. The nanoparticles could be prepared only within a zone of appropriate chitosan and TPP concentrations. The particle size and surface zeta potential can be manipulated by variation of the fabrication conditions such as chitosan/TPP ratio and concentration, solution pH and salt addition. TEM observation revealed a core–shell structure for the as-prepared nanoparticles, but a filled structure for the ciprofloxacin (CH) loaded particles. Results show that the chitosan nanoparticles were rather stable and no cytotoxicity of the chitosan nanoparticles was found in an in vitro cell culture experiment. Loading and release of CH can be modulated by the environmental factors such as solution pH and medium quality. Copyright © 2008 John Wiley & Sons, Ltd.
Article
Hydrophilic nanoparticulate carriers have important potential applications for the administration of therapeutic molecules. The recently developed hydrophobic-hydrophilic carriers require the use of organic solvents for their preparation and have a limited protein-loading capacity. To address these limitations a new approach for the preparation of nanoparticles made solely of hydrophilic polymers is presented. The preparation technique, based on an ionic gelation process, is extremely mild and involves the mixture of two aqueous phases at room temperature. One phase contains the polysaccharide chitosan (CS) and a diblock copolymer of ethylene oxide and propylene oxide (PEO-PPO) and, the other, contains the polyanion sodium tripolyphosphate (TPP). Size (200–1000 nm) and zeta potential (between +20 mV and +60 mV) of nanoparticles can be conveniently modulated by varying the ratio CS/PEO-PPO. Furthermore, using bovine serum albumin (BSA) as a model protein it was shown that these new nanoparticles have a great protein loading capacity (entrapment efficiency up to 80% of the protein) and provide a continuous release of the entrapped protein for up to 1 week. © 1997 John Wiley & Sons, Inc.
Article
The application of nutraceuticals in the field of male sexual function -in particular for erectile dysfunction (ED)--remains relatively underexplored. In a group of 54 unselected men (35-75 years), consecutively presenting to our ED clinic and naive to other ED treatments, we carried out a single-blind, one-arm study to evaluate the effects of a 3-month supplementation with propionyl-L-carnitine, L-arginine and niacin on their sexual performance. All patients had the short-international index of erectile function (IIEF) questionnaire, global assessment questions (GAQs) and routine laboratory testing, at baseline and 3 months afterward. 51 (92%) patients of 54 completed the entire study period. After 3 months of treatment, a small, but statistically significant improvement in total and single items of the IIEF was found (Δ = 5.7 ± 4.1 P < 0.01). Analyses on GAQs revealed that treatment improved erections in 40% of cases, with a partial response occurring in up to 77% of subjects enrolled. These preliminary findings indicate that the favourable cardiovascular effects of nutraceuticals might also reflect on male sexual function with possible implication in the treatment and prevention of ED. This study documents a considerable patient's interest toward nutritional supplementation--as first-line or adjunctive treatment to PDE5 inhibitors--that goes beyond the measurable increment in penile rigidity.