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Copyright 2011 American Scientic Publishers
All rights reserved
Printed in the United States of America
Journal of
Bionanoscience
Vol. 5, 155161, 2011
Layer-by-Layer Self-Assembly of Polymeric
Multi-Layers on Solid Lipid Nanoparticles:
A Comparative Study via Dynamic Light
Scattering, Transmission Electron Microscope,
Atomic Force Microscope and Quartz Crystal
Microbalance with Dissipation
Thiru G. Ramasamy
1
and Ziyad S. Haidar
12
1
Department of Bioengineering and Regenerative Medicine, Utah-Inha DDS and Advanced Therapeutics Research Center,
B-404 Meet-You-All Tower, Songdo TechnoPark 7-50, Songdo-Dong,
Yeonsu-Gu, Incheon, 406-840, Republic of South Korea
2
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah,
30 South 2000 East, Salt Lake City, UT, 84112-5820, USA
SLN or solid lipid nanoparticles were suggested as advantageous alternatives to liposomes and
other colloidal carriers for hydrophobic drug delivery, yet with limited loading capabilities and dimin-
ished tunable release proles, especially for hydrophilic proteins and peptides. Thus, we pro-
posed formulating core-shell nanocapsules: coating SLN cores with alternate layers of natural
polyelectrolytes via the layer-by-layer self-assembly approach based on electrostatic interactions.
In the present work, tricaprin-based SLN were simply produced, a bi-layered shell constructed and
step-wise characterization carried out by DLS, TEM and AFM. To further gain an understanding,
we employed quartz crystal microbalance with dissipation (QCM-D) to monitor and compare, the
build-up, in real time. Findings reveal that alginate-chitosan ([ALCH]) and hyaluronan-chitosan
([HACH]) layers were successfully deposited on SLN. QCM-D provided further evidence that poly-
electrolyte shells range 4060 nm in thickness with differences attributed to dissimilar growth
regimens in both systems. A viscous yet compact and rigid structure was formed in the AL-based
system while a oppy and spongy multi-layer was formed with HA. To the best of our knowledge,
this is the rst report depicting the formation and characterization of polyelectrolyte multi-layers on
SLN and analyzing structural properties of core-shell colloids on a gold surface by QCM-D tool.
Keywords: Alginate, Chitosan, Dissipation, Frequency, Hyaluronic Acid, QCM-D, Solid Lipid
Nanoparticles.
1. INTRODUCTION
Almost two decades passed since Hong and Decher
1
rst
introduced the basic principle of versatile polyelectrolyte
multilayers or PEM formed via the layer-by-layer (l-b-l)
self-assembly of oppositely-charged materials. This sim-
ple and malleable technology has found growing appli-
cations ever since, especially in the coating and surface
functionalizing (or tuning) of colloidal particles, thin opti-
cal lms, sensors, electronic devices and denitely so in

Author to whom correspondence should be addressed.

controlled drug and therapeutic agent delivery.
24
Indeed,
our own works demonstrated how such thin PEM lms
of supramolecular architecture formulated via the l-b-l
technique (based on electrostatic interactions) surround-
ing liposomal cores greatly facilitated, while maintaining
bioactivity, incorporation and release prole modulation of
bone growth factors with promising applications in tissue
engineering and regenerative medicine.
57
For most PEM
applications, natural polymers such as anionic alginate
(AL), hyaluronic acid (HA) and cationic chitosan (CH),
which have been playing an important role in biomedical
J. Bionanosci. 2011, Vol. 5, No. 2 1557-7910/2011/5/155/007 doi:10.1166/jbns.2011.1058 155
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Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles Ramasamy and Haidar
research, have been employed. Besides their attractive
biocompatibility, biodegradability and ease of processing,
these polyelectrolytes have potential to mimic some bio-
logical environments.
611
However, not much is known on
the structural properties of these PEM. Furthermore, lipo-
somal vesicles tend to suffer from stability problems due
to fragility, given the signicant mass of adsorbed PEM.
PEM, assembled on top of nanoparticles serve as barri-
ers (shell) for drug diffusion, thus allowing the control
of load release.
12
This shell composition and thickness
also dictates the degradation/erosion speed as well, further
facilitating release modulation. Attractive colloidal drug
carrier alternatives to liposomes, solid lipid nanoparticles
or SLN, which are natural/physiological lipids, have been
suggested. This is mainly due to their long-term stability,
biocompatibility, scalability, sterilizability and feasibility
of delivering both lipophilic as well as hydrophilic drugs.
13
Nevertheless, the main drawback of SLN and SLN-based
systems continues to be the limited loading capability
14
and consequently difculties in tailoring release proles.
Hence, we attempted to combine the advantages of SLN
with those of PEM via the application of the l-b-l self-
assembly of alternating layers of polyelectrolytes to for-
mulate stable nanocapsules with increased encapsulation
capacity/efciency and release modulation via control-
ling the number/thickness of the constructed shell. Herein,
biodegradable pairs of AL and CH or HA and CH were
assembled onto cationic SLN. Our aim is to (a) assess the
feasibility and benets of PEM on SLN and more impor-
tantly to (b) understand differences between [ALCH]
2
and [HACH]
2
PEM systems. To the best of our knowl-
edge, this is the rst report to demonstrate a PEM shell
build-up on SLN core. Additionally, no studies compare
the growth regimens of [ALCH] and [HACH] PEM sys-
tems, whether on at planar surfaces or colloidal parti-
cles. This comparative study to understand the structural
and visco-elastic differences of the l-b-l build-up was car-
ried out in situ by means of resonant frequency (A] )
and energy dissipation (AD) measurements obtained via
the quartz crystal microbalance with dissipation (QCM-D)
tool, step-wise and in real time. PEM visco-elastic prop-
erties are crucial for biomedical applications inuencing
cell/biomaterial interactions and drug, protein or agent
incorporation and release kinetic.
2. EXPERIMENTAL DETAILS
2.1. Materials
Tricaprin (Tc) main lipid was purchased from Sigma-
Aldrich Chemical. 3p-[N-(N

,N

-dimethylaminoethane)-
carbamoyl]cholesterol hydrochloride (DC-Chol),
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE)
were purchased from Avanti

Polar Lipids, South Korea.

Tween 80, sodium alginate (low molecular weight, vis-
cosity: 250 cps), chitosan (medium molecular weight,
viscosity: 200 cps, degree of deacetylation: 85%) and
hyaluronic acid (low molecular weight, viscosity: 250 cps)
were all purchased from Sigma-Aldrich Chemical. For the
l-b-l build-up, fresh polymer solutions were prepared in
ultra-pure water (18.2 MD cm
1
) with a nal concentra-
tion of 1 mg.mL
1
. The pH of CH was maintained at 5.5
while HA and AL at pH 7.0 using glacial acetic acid and
sodium hydroxide solutions (Sigma-Aldrich Chemical),
respectively. The gold (Au) QSX-301surface (100 nm
Q-Sense

sensor) was purchased from Biolin Q-Sense

,
Sweden.
2.2. Formulations: a Step-Wise Characterization of
SLN[ALCH]
2
and SLN[HACH]
2
NCs
SLN were prepared via the melt homogenization method
with slight modication.
15
Briey, Tc (30 mg), DOPE
(9 mg), DC-Chol (9 mg) and Tween 80 (0.2 mL) were dis-
solved in 3 mL of tertiary butyl alcohol. Following rapid
freezing in a liquid nitrogen tank, mixtures were dried
overnight in a lyophilizer (Labconco, Missouri, USA).
The obtained nely-dispersed cakes were then placed in
a water bath maintained at 50

C. Homogenization for
5 at 11000 rpm using a high pressure homogenizer
(ULTRA-TURRAX T25 basic, Germany) and sonication
in a probe type sonicator (SONICS, Vibra cell, USA) set
at 60% amplitude for 5 followed, resulting in a turbid-
white emulsion. The formulated SLN were then charac-
terized by dynamic light scattering (DLS) using a high
performance zeta sizer (ZS 90, Malvern instruments, Ger-
many). SLN morphology was studied using a transmission
electron microscope (TEM, CM 200 UT, Philips, USA)
where about a 10 jL suspension sample was placed on
a 300-mesh copper holey carbon grid and stained with
2% w/v phosphotungstic acid (PTA). Following air dry-
ing, visualization was carried out at 120 kV. Likewise,
surface topography was studied by an atomic force micro-
scope (AFM, NanoFocus, from Digital Instruments, USA)
in contact mode using a cantilever of silicon probe type
with a resonance frequency of about 300 kHz. Height
proles (from surface) were deduced using the accom-
panying WSxMImage Browser software. The l-b-l self-
assembly of polymers
57
followed as we reported earlier.
Briey, cationic SLN were coated with alternating lay-
ers of anionic AL or anionic HA and cationic CH (vol-
ume ratio of 1:2, respectively) until a double bi-layered
shell (4 layers) was formed around the lipid core. With
the deposition of each polymeric layer, the nanocapsules
(NCs) solution was incubated at RT for 60 under gen-
tle stirring. Centrifugation at 1800 rpm for 15 to elimi-
nate aggregates that may form upon the mixture of SLN
and the polymeric material (washing) followed. Char-
acterization by DLS, TEM and AFM was also done,
step-wise.
156 J. Bionanosci. 5, 155161, 2011
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Ramasamy and Haidar Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles
2.3. QCM-D: Monitoring the Shell l-b-l Build-Up in
Real Time on Au and SLN/Au Surfaces
QCM-D measurements were performed using the Q-
Sense

E
1
system equipped with a QFM 401 Q-Sense

ow module (for 5 MHz QSX) via monitoring simultane-

ously the changes in frequencies (] ) and dissipation (D)
following each material deposition step. An AT-cut quartz
crystal oscillator (14 mm in diameter and a fundamental
frequency of 5 MHz was used. A clean 100 nm Au-coated
sensor was mounted into the liquid exchange chamber of
the instrument and a baseline initially established. Temper-
ature was kept at 240.05

C throughout all experiments.
Ultra-pure water (18.2 MD cm
1
) was passed rst for
10 min, after which polyelectrolyte solutions were injected
into the measurement cell for a period of 15 min and at a
ow rate of 50 jL/min using a peristaltic pump. A 5 min
rinsing step with ultra-pure water was performed between
the adsorption of each polyelectrolyte material. Qtools,
a Q-Sense

Analysis software (Q-Sense

AB) was used

to model the obtained results. Changes in resonance fre-
quency (A] ) and energy dissipation (AD) for the 3rd, 5th,
7th and 9th overtones were tted according to the Voigit-
based model
17
to analyze the thickness and viscosity of the
adsorbed material. The following parameters were xed
throughout the study: bulk liquid density: 10
3
kg m
3
; bulk
liquid viscosity: 10
3
kg m
1
s
1
; adlayer density: 1.1
10
3
kgm
3
. A total of 3 runs was performed comparing
the adsorption of (a) [ALCH]
2
to [HACH]
2
on Au and
(b) [ALCH]
2
to [HACH]
2
on SLN previously applied
on the Au surface.
3. RESULTS AND DISCUSSION
We are interested in formulating a stable and tunable
SLN-based delivery system for potential applications in
skin tissue engineering and regeneration, given the GRAS
(Generally Recognized as Safe) status for SLN as they are
physiological lipids. Herein, SLN were simply produced
Fig. 1. Schematic presentation of the different materials used in this study and an illustration of the layer-by-layer build-up of polyelectrolytes on
tricaprin SLN/Au surface.
by the melt homogenization technique using tricaprin, a
lipid with short fatty acid (carbon) chain length (Fig. 1).
Tricaprin was selected to produce compact SLN. Char-
acterization data are displayed in Figure 2. DLS reports
SLN with an average hydrodynamic diameter of 180
2.8 nm and surface charge in the order of +38 mV.
More importantly, the l-b-l self-assembly of polymers
succeeded in constructing a double bi-layered shell on
SLN cores. This is evident from the changes in particle
size and surface charge. SLN[ALCH]
2
and SLN [HA
CH]
2
had a cumulative average size of 380 nm and a
nal (outermost CH layer) surface charge in the order of
+32 mV. As expected from our previous works,
59
the
adsorption of each polymeric layer resulted in an increase
in size and a surface charge inversion. It is noteworthy
that SLN [ALCH]
2
are smaller nanocapsules (NCs) than
SLN [HACH]
2
. Zeta potential is the potential within the
hydrodynamic shear or slipping plane of the electric dou-
ble layer of a charged particle. A stable -potential (val-
ues above +30 mV or below 30 mV) manifests that
no aggregation occurred during the l-b-l process. Oth-
erwise, the mobility of the colloid particle would have
decreased, resulting in a decrease in -potential. Further,
the alternation in surface charge polarity following each
deposition is necessary for the l-b-l assembly of oppo-
sitely charged polyelectrolytes.
17
Hence, our ndings con-
rm the presence and coverage of the polymer coating
and indicates the stability of the formulated NCs.
15
TEM
morphological views further conrm DLS ndings reveal-
ing a darker PTA-stained SLN surrounded by a grayish
polymeric shell. No clear distinction between the different
polymer layers was feasible, due to the close/longitudinal
attachment (dense complexation) of the polymer chains,
however, monodispersity retained. AFM provided evidence
on the stability of the formulated carriers (withstanding the
multi-dimensional scanning forces); preserving their spher-
ical morphology; and presence of the polymeric shells.
Height proles suggest the differences in overall parti-
cle size; attributed to the structural properties of the shell
J. Bionanosci 5, 155161, 2011 157
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Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles Ramasamy and Haidar
Fig. 2. (a) Changes in average hydrodynamic diameter, -potential with the adsorption of each of each polymeric layer on SLN. (b) TEM images
(Bar = 500 nm) of SLN and coated SLN showing effect of polymeric shell and the polydispersity index (PDI). (c) AFM micrographs illustrating
surface topographies of uncoated SLN, ALCH coated SLN and HACH coated SLN with their relevant height proles (distance from surface).
where the larger SLN [HACH]
2
were signicantly thicker
(j - 0.05) than the smaller and thinner SLN [HACH]
2
;
predicting that the former system would possess a much
higher drug loading capacity than the latter. Collectively,
monodisperse and stable SLN and coated SLN are evi-
dent, setting those novel NCs superior to our previously
formulated liposome-based nanoparticulate drug/protein
delivery system.
57
The polydispersity index (PDI) is a
measure of dispersion homogeneity ranging from 0 (homo-
geneous) to 1 (heterogeneous).
5, 6
PDI values were lower
than 0.175 for uncoated SLN, suggesting a relatively nar-
row size distribution and production method suitability.
While PDI increased amid polymer addition, neverthe-
less, values remained in the range specifying uniformity;
directly proportional to NC size changes as reported via
DLS, TEM and AFM.
To further understand the assembled layers, QCM-
D was employed to study the growth regimens of the
shell, directly and indirectly (on the Au sensor). Figure 3
displays the obtained plots of frequency (] ) and dissipa-
tion (D) changes measured in real-time, monitoring the
build-up of [ALCH] and [HACH] double bi-layered
shells on SLN cores. For this purpose, CH solution (at
pH 5.5) and HA and AL solution (at pH 7.0) were pre-
pared. Since polyelectrolyte deposition is primarily gov-
erned by electrostatic interactions, the degree of ioniza-
tion of the charged molecule is of prime importance.
18
Charge density and conformation of weak polyelectrolytes
is highly inuenced by changes in pH.
16
Therefore, the pH
of CH (Ko =6.6) was adjusted to 5.5 where charge den-
sity is increased due to the protonation of amine groups
facilitating interactions with carboxylate groups of AL and
HA.
18
Conversely, the pH of AL and HA (Ko =3.0/4.5)
was adjusted to 7.0, well above their pKa, to obtain max-
imum charge density.
17
It is noteworthy that the upper
curves in Figure 3 denote the changes in frequency (A] )
and the lower curves show the measured changes in dis-
sipation (AD) over time. As expected, the adsorption of
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Ramasamy and Haidar Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles
Fig. 3. Plots of QCM-D data showing the frequency and dissipation shifts as a function of time (3rd, 5th, 7th, and 9th overtones) for the HACH and
ALCH bi-layers on SLN. Start of each adsorption steps is marked with an arrow (). Intervals: alternating 5 min (rinse) and 25 min (adsorption) steps.
each polyelectrolyte layer can be distinguished step-wise
where each step corresponds to a decrease in ] and an
increase in D, indicating that mass is being deposited onto
the crystal surface; exhibiting certain visco-elastic proper-
ties. It can also be clearly observed that the decrease in ]
for all 4 overtones is different and at no point they over-
lap. This indicates that the adsorbed layer does not obey
the Sauerbrey equation,
19
attributed to many reasons such
as the visco-elastic behavior of the adsorbed lm and that
the lm may not be homogeneously-deposited on the sur-
face. Primarily, cationic SLN were adsorbed to the bare
Au surface resulting in an initial ] shift of 2933 Hz. SLN
seems to have a strong afnity to Au demonstrated by the
steep one time decrease in ] , quickly saturating afterwards
indicated by a at ] curve until polymer layers were intro-
duced. This further implies the integrity of SLN within
the assembly, consistent with the DLS, TEM and AFM
results. QCM-D reveals different growth patterns associ-
ated with each polymeric self-assembled system. With the
rst layer of AL or HA, a decrease in ] was observed,
yet, with key variances. Cationic SLN are compact, so we
anticipated a strong interaction with the negatively-charged
AL and HA, likely to cause domain construction. Subse-
quent assembly of the rst cationic CH layer on both sys-
tems (SLN-AL and SLN-HA) induced a prominent shift of
nearly 50 Hz. Likewise, after the last CH layer, ] shifted to
100 Hz in the HA-based system and 70 Hz in AL, indicat-
ing that maximum mass increase is indeed attributed to CH
which in turn is due, again, to the high swelling and water
incorporation phenomena. Such differences in energy dis-
sipation are related to the visco-elastic properties of the
deposited PEM shell or lm. In real time, when a poly-
mer adsorbs to the nanoparticle surface or on the top of
another polymer, water couples to the adsorbed polymer as
an additional mass. Therefore, the polymer lm is sensed
by the QCM-D instrument as a hydrogel. Furthermore, D
determines if the lm/hydrogel is rigid or elastic. More
importantly, the amount of H
2
O adsorbed within a lm is
dependent on the polymer intrinsic properties
16
measured.
In this regard, for the rst layer of HA deposited on the
SLN, D changes to 11 10
6
compared to only 5.8
10
6
for AL (3rd overtone) hinting that HA is far more
visco-elastic than AL. However, subsequent CH adsorp-
tion produced a similar yet larger D hinting its bulki-
ness. Overall, both systems showed an elevated D, with
signicantly (j - 0.05) higher values for [HACH] than
[ALCH], rendering the former more visco-elastic than
the latter. Further, AL chains usually take more extended
conformation at lower ionic strength due to the electro-
static repulsion, which, in turn, causes a thinner assembled
multi-layer.
13
This compact layer structure has less free
space or voids for solvent to be trapped within explaining
the lower relative solvent (H
2
O) content in AL-based sys-
tems, further accounting for the low D value.
14
Another
notable observation is that after HA adsorption, initially
there was a high D (and high ] ) which is followed by
gradual decrease in energy D. A possible explanation is
the protruding and penetration/diffusion of HA into the
previously adsorbed CH layer.
20
Thus, the major channel
for energy loss of the oscillating sensor is not the vis-
cous deformation of the shell alone, but the energy D to
the environment through mechanical coupling of the out-
ermost part of the multi-layer to the liquid phase (or top
layer). The Viogit-based method
16
was used to model the
thickness of both systems. [HACH] grew to 62 nm while
[ALCH] was thinner at 41 nm, corresponding to the pre-
viously described DLS and AFM ndings. Film thickness
increased with an average of 12.6 nm for HA and 8 nm for
AL, per layer. This further implies that the structure of the
formed PEM is non-rigid, oppier and spongy with HA
than the more compact and rigid AL-based system. This
is in agreement with the work of Salomaki and Kankare
21
who suggested that the build-up regimen is related in part
to the resultant stiffness of a polymer lm, with expo-
nentially growing lms considered softer than lms with
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Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles Ramasamy and Haidar
Fig. 4. Differences (a) thickness and (b) sensed mass per layer adsorbed (on SLN/Au) comparing AL-CH and HACH PEM build-up. (c) Comparative
structural and visco-elastic properties between ALCH and HACH ci-layers adsorbed on Au and those adsorbed on SLN, by QCM-D.
linear growth. Hillberg and others
14
also demonstrated an
exponential growth for the [HACH] regimen and a linear
growth for [ALCH] on at surfaces. Linear growth, how-
ever, was driven by the over-compensation of the surface
charge following each polyelectrolyte adsorption leading
to limited interpenetration of neighboring layers.
22
On the
other hand, exponential growth was attributed to the ability
of HA or CH to diffuse through the shell structure. In the
latter, the lm constitutes a reservoir of free chains,
which can diffuse-out and immobilize an excess amount of
oppositely-charged polyelectrolytes during the following
adsorption step (restricted mobility of the polyelectrolytes
within the multi-layer).
23
To differentiate between deposi-
tion of polymers on a colloidal substrate or a at surface,
we repeated a similar l-b-l build-up on a plain Au surface.
Findings are tabulated (insert) of Figure 4. As expected,
] and D shifts for polymer deposition on a at surface
were signicantly (j -0.05) lower than on a surface with
colloidal particles. To be specic, sensed mass follow-
ing the adsorption of the rst layer measured at 1200
1600 ng/cm
2
on colloids, while only 700780 ng/cm
2
on the at surface (previously stabilized by PEI). Nev-
ertheless, similar growth patterns were evident, implying
that the structural conformation of SLN allowed a higher
polymeric mass uptake. From the D proles, it is obvi-
ous that the adsorbed amount, and thus the sensed mass,
substantially increased with the adsorption of CH, sug-
gesting that CH behaves as a more visco-elastic hydrogel
due to swelling (more mass on sensor). D data can further
be split into viscous and elastic components (Voigit). The
viscosity/shear modulus components were relatively low
after the rst adsorbed layer of HA and AL, conversely so,
following the adsorption of CH, where a substantial and
instantaneous increase was observed. This trend contin-
ues with increasing layer number and with a signicantly
higher viscosity/shear modulus for the HA-based system.
As indicated in the plots of Figure 4, higher mass was
deposited with the HA-based system, evident with a value
of 1250 ng cm
2
compared to the 800 ng cm
2
mass
with AL. It is therefore most likely that HA is more visco-
elastic on top of the previous CH layer that is essentially
rigid. The adsorbed amount of polyelectrolyte includes a
substantial contribution from the entrapped solvent
23
in
which the polymer is dissolved. Finally, the importance of
this study lies in that thick and visco-elastic (gel-like) char-
acteristics are directly related to the swelling and hydrat-
ing properties of lms, a way to predict cell adhesion.
Poor adhesion results from cells identifying the surface
as water, a concept previously suggested by Mendelsohn
et al.
24
In addition, lm stiffness is a key parameter to
consider when designing a surface that promotes cellular
adhesion.
10
Protein adsorption, given the possible applica-
tion of NCs for controlled and tunable drug delivery, is
also dependant on the nature of the PEM shell formed.
25
Ongoing works attempt to evaluate/quantify protein load-
ing and encapsulation efciency as well as kinetics and
bioactivity of released drugs from PEM lms and core
shell NCs.
4. CONCLUSION
To the best of our knowledge, this is the rst report on
PEM shell build-up via the l-b-l technique on a SLN core,
denoting different growth regimens of [ALCH] and [HA
CH] systems. The physico-chemical properties of PEM
were investigated by means of DLS, TEM, AFM and
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Ramasamy and Haidar Layer-by-Layer Self-Assembly of Polymeric Multi-Layers on Solid Lipid Nanoparticles
QCM-D. Our ndings demonstrate that HA-based systems
grow exponentially to a thickness of 63 nm, while AL-
based systems grow linearly to 41 nm on SLN. A viscous
yet compact and rigid structure was formed in the lat-
ter compared to a more open, oppy and spongy (water
rich) shell formed in the former. Such ndings open new
doors to formulating multi-layered polymeric shells on
SLN and other cores, with potential applications in bio-
physical/-medical elds. To render our nanocapsules truly
generic, cell-/-protein interaction studies are vitala sub-
ject of ongoing research.
Acknowledgments: This work was supported by the
South Korean Ministry of Knowledge and Education
(MKE) and the Incheon Free Economic Zone (IFEZ) in
the framework of several funding operating grants to the
Utah-Inha DDS & Advanced Therapeutic Research Cen-
ter, KR.
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(2008).
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Received: 16 December 2011. Accepted: 1 February 2012.
J. Bionanosci 5, 155161, 2011 161