Transcript Blg

Chitosan/B-lactoglobulin core-shell nanoparticles as
nutraceutical carriers
Presenter: Jeong Youngjin
Background
Tries to develop innovative functional foods that may have physiological benefits or
reduce the risks of diseases to improve public health.
The incorporation of bioactive compounds—such as peptide, vitamin—into food
systems as a potentially simple means of modulating the risks of diseases.
However, The effectiveness of such products relies on preserving the bioavailability of
the active ingredients.
remain poorly available by oral administration due to too short gastric residence time of
the dosage form; low permeability and /or solubility within the gut; lack of stability in
the environmental conditions encountered in food processes (temperature, oxygen, light)
or in the gastro-intestinal tract (pH, enzymes, presence of other nutrients), which limits
their activity and potential health benefits.
Encapsulation systems can be used to overcome these limitations.
The growing interest in the effective and selective delivery of bioactive agents to the
site of action has led to the development of new encapsulation materials.
Chitosan
Chitosan (CS), a copolymer derived from chitin, is a natural nontoxic biopolymer derived
from fully or partially deacetylation of chitin, which is structural element in the
exoskeleton of crustaceans(crabs, shrimp, etc). CS has biocompatible, biodegradable and
antimicrobial properties.
CS is allow to soluble at low pH(6.5) aqueous solution and tends to be positively
charged, thus can be combined with negatively charged TPP
These properties make CS a good candidate for the development of nutraceutical
delivery systems for food applications. However, for oral administration, CS matrix are
not stable at low pH values, and rapid dissociation and degradation occurred at pH 1.0,
which could lead to destruction of sensitive nutraceuticals in the stomach circumstances.
To overcome the drawbacks, the CS nanoparticles could be surface coated to enable the
protection in the gastric tract.
B-lactoglobulin (Blg)
B-lactoglobulin, the major wheyprotein in the milk, is a small globular protein widely
used as food ingredient because of its nutritional value and its ability to form gels,
emulsions, and gelled emulsion.
Important functional property of whey proteins
-Ability to form cold induced gel matrices by adding cations to a preheated denatured
protein suspension, which results in the formation of a network crosslinked via Ca2+ with
carboxylate groups on denatured Blg at ambient temperature.
-It is known to be resistant to degradation of the pepsin in the stomach in its native
structure or adsorbed at an interface. Hydrolysis of whey proteins by pancreatin
enzymes generate bioactive peptides that may exert a number of physiological effects
in vivo, e.g. on the gastrointestinal, cardiovascular, endocrine, immune and nervous
systems.
Due to its attractive techno-functional properties and large availability Blg is an
interesting candidate to coat CS nanoparticle to allow a protection when subject to the
gastric fluids.
Preparation of native and denatured blg solutions
Native Blg
Hydrating Blg in deionized water
Agitation at room temperature for 1h
Denatured Blg
Negligence for 2 h
Heat at 80 ℃ and 2.4 for 30 min
Formation of CS-Blg nanoparticles
1. Native or denatured Blg solutions at various concentrations and pH values were
added to CS solutions in aqueous acetic acid (0.1%) to form CS-Blg complexes with CS
concentration of 2.0 mg/ml.
2. Then TPP was dissolved in distilled water at 1.0 mg/ml. Finally, 2 ml of TPP solution
was added dropwise to 5ml of CS-Blg complexes, opalescent suspension was formed
spontaneously under magnetic stirring at room temperature, and was further examined
as nanoparticles.
3. The final pH of the nanoparticle suspension was measured with an Orion 370 pH
meter (Orion Research, Inc. MA), and the nanoparticles were characterized immediately.
All experiments were performed in triplicates
Influence of the pH value on the nanoparticle properties
Measurements of particle size and zeta potential of the nanoparticles were performed
with photon correlation spectroscopy and laser Doppler Anemometry, using a
Mastersizer 2000 and Zetasizer 2000, respectively (Malvern Instruments, South borough,
MA). The size measurements were performed at 25 ℃ and at a 90 ℃ scattering angle. It
was recorded for 180 s for each measurement. The mean hydrodynamic diameter was
generated by cumulative analysis. The zeta potential measurements were performed
using an aqueous dip cell in the automatic mode.
Measurements of particle size and zeta potential of the nanoparticles were performed
with photon correlation spectroscopy and laser Doppler Anemometry, using a
Mastersizer 2000 and Zetasizer 2000, respectively (Malvern Instruments, South borough,
이온과
입자가
안정하게
존재하는
이론적인
경계.
MA). The size measurements were performed at
25 ℃
and at
a 90 ℃
scattering
angle.
It
제타포텐셜의
크기는
coloidal
시스템의
포텐셜적인
was recordedAt
forlow
180
for each
measurement.
Theamino
meangroups
hydrodynamic
diameter
was
pHs value
(pH<3),
majority of the
(over 90%)
are protonated
안정성을
나타냄.
Suspension에
있는
모든
입자가
form an extended
in themeasurements
acid solution due
to strong
repulsion
generated byto
cumulative
analysis.molecular
The zetachain
potential
were
performed
큰 음전하
혹은Therefore
양전하의 제타전위를
가지고 있을
existing
charged
amino
groups.
a more extended
using an aqueous
dipamong
cell inpositively
the automatic
mode.
때 서로 많이 반발하는 경향을 가지게 되고 이들은
spherical shape is formed upon addition of the TPP solution. While with increase
서로 결합 하려 하지 않는다. 그러나 낮은 제타전위
of the pH value (pH 3–9), positive charges would be neutralized with gradual
를 가지게 될 경우 입자들이 서로 반발하는 힘이 줄
deprotonation of the amino groups, resulting in a less extended molecular chain
어 들게 되고 결국 응집이 일어나게 된다.
of CS to form uniform nanoparticles with small size.
일반적으로 안정하거나 불안정한 suspension을 나
누는 기준은 +30 or – 30 mV이다.
CS-Blg nanoparticles
TEM photographs (Fig. 1(a)) show Surface and
interior morphology of CS-Blg nanoparticles
prepared with native Blg at pH 6.1 with CBlg of 2.0
mg/ml.
Fig. 1(b) show the particle size about 100 nm,
indicating that nanoparticles are formed—they
appear spherical in shape with smooth surfaces.
The interior structure of CS-Blg nanoparticles
demonstrates a circular shape consisting of a dark
core and a light shell.
Fig. 1. Surface and interior morphologyof CS-blg nanoparticles
prepared with native blg at pH 6.1 with Cblg of 2.0 mg/ml.
Blg coating properties
To determine the association efficiency(AE) and loading efficiency(LE) of Blg on the
nanoparticles, triplicate batches of nanoparticles were centrifuged at 30,000g (RC5C
Sovall Instruments Dupont, Newton, Conn.), 20 ℃ for 30 min, and the Blg content in the
supernatant was determined by UV spectrophotometry (HP 8453 UV–Visible,
spectrophotometer, Palo alto, CA) at 280 nm. The pellet was vacum dried and weighted.
The AE and LE values were calculated by the following formulae:
Influence of the pH value on the nanoparticle properties
63.4% at pH 7.1
↓
42.8% at pH 6.1
↓
4.3
5.3
5.9
The effects of pH values on AE and LE of Blg on CS-TPP core are
shown in Fig. 2. Three regions with two transition points at pH
4.3 and 5.9 in the pH range used were observed for both native
and denatured Blg.
-pH value < 4.3
A small amount of blg were coated on CS-TPP core.
Both CS and Blg are positively charged, strong repulsion
prevents association of Blg on CS-TPP core.
-4.3 < pH value < 5.9
pH value was higher than 4.3, AE values for both native and
denatured blg began to increase steadily
The pI of Blg exists, intraionic attractions between COO- and
NH3 + result in seldom residual ionic groups on Blg. In this pH
range, hydrophobic interactions and hydrogen bondings between
Blg and CS are supposed to dominate to explain the steadily
increase of the AE value
-pH value > 5.9
AE value increased sharply to a maximum of 42.8% for native blg
at pH 6.1 and 63.4% for denatured blg at pH 7.1. CS is positivelyc
harged, while Blg becomes negatively charged, the driving force
for Blg association thus changed from hydrophobic interactions
gradually to electrostatic attractions
Influence of the pH value on the nanoparticle properties
The LE values which measure the amount of Blg loaded on unit
weight of nanoparticles are also strongly pH dependent and
showed similar changing tendency of AE values with two
transition points as a function of pH, as demonstrated in Fig. 2(b),
4.3
5.3
5.9
pH 6.1
↓
pH 6.5
↓
Confirming change of interactions between CS and Blg at
different pH values. However, the maximum LE values were
obtained at pH 6.1 for native Blg and pH 6.5 for denatured blg,
and with further increase of the pH, LE values for both native and
denatured Blg decreased. A reasonable explanation for this
decrease could be conformation change of the absorbed Blg.
When denatured by heating, Blg molecular chains tend to be
random coils. Near pI, while with the remaining hydrophobic
portion of the molecule lying close to the surface. Thus steric
hindrance between proteins prevents further adsorption of the
denatured Blg. Therefore, one unit weight of CS-nano allows
more adsorption of the native Blg, which leads to a relatively
high LE value
The LE value of native Blg loaded on unit weight of CS-TPP core
are much higher than those of denatured Blg in all pH values
investigated in this study, as obviously shown in Fig 2(b).
Influence of Blg initial concentration on the nanoparticle properties
Native Blg
Denatured Blg
Table 2 depicts the effects of initial Blg concentrations (CBlg) on nanoparticle properties
at various pH values. No obvious differences in the mean diameter, polydispersity and
zeta potential among CS-Blg nanoparticles prepared with different CBlg at any specified
pH value were observed, indicating that Cblg does not play an important role in
regulating particle size, size distribution and surface charges of the CS-blg nanoparticles.
Brilliant blue release
In order to investigate the feasibility of CS-Blg nanoparticles as carriers for nutraceuticals. In
vitro release properties of BB into simulated gastric-intestinal tract were evaluated for CSBlg
nanoparticles formed with a CBlg of 2.0 mg/ml at pH 6.1 for native Blg and pH 6.5 for
denatured Blg (Labeled as nanonative and nanodenatured). Nanoparticles prepared with
denatured Blg was also crosslinked with CaCl2 (10 mmol/l) over night to form a network with
formation between carboxylate groups of denatured Blg and Ca2+ (Labeled as
nanocrosslinked). This work was intended to obtain a optimal shell structure of the
nanoparticles.
Brilliant blue (BB) was used as model molecules. Since BB is a small molecule with high
anionic charge densities, strong electrostatic interactions between BB and amino groups of CS
played a very important role in BB encapsulation.
The molecules encapsulated nanoparticles were prepared by incorporating BB into the CS-Blg
complexes to a final concentration of 0.2 mg/ml, prior to the formation of the nanoparticles.
For the determination of the drug encapsulation capacity, the BB encapsulated CS-Blg
nanoparticles were separated from the aqueous suspension medium by ultracentrifugation
with 30,000g at 20 ℃ for 30 min. The amount of free BB in the clear supernatant was
determined by measuring the UV–vis absorption at 570nm (HP 8453 UV–Visible,
spectrophotometer, Palo alto, CA). BB encapsulation capacity(EC) was calculated with the
following equation:
Release test to target organ
pH 1.2
↓
pH 7.5
↓
With pepsin
With pepsin and
pancreatin
Without enzyme at pH 1.2
Conclusion
This work has elaborated CS-Blg core–shell nanoparticles by cold ionic gelation of
chitosan (CS) and Blg mixtures with TPP. The coating properties of native and denatured
Blg on CS-TPP core were highly sensitive to formulation pH and CBlg. Optimal
nanoparticles with size of about 100nm were obtained at pH 6.1 for native
Blg and pH 6.5 for denatured Blg at CBlg of 2.0 mg/ml, where the native Blg exhibits an
end-on conformation and denatured Blg forms a tail projected from the positively
charged surface with both of their hydrophobic portions of the molecule lying close to
the CS-TPP core to allow a maximum Blg loading. The BB release experiments indicate
that Blg in its native formation is an interesting candidate to coat CS nanoparticle to
allow a protection of nutraceutical coumpounds when subjected to the gastric fluids
due to favorable properties to resist acid and pepsin degradation in the gastric fluids.
When transferred to the intestinal fluids, the outside shells of these nanoparticles were
degraded by pancreat.