European Journal of Pharmaceutical Sciences 105 (2017) 55–63

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Preparation of liposomes containing small gold nanoparticles using MARK

electrostatic interactions
Gennaro A. Dichelloa, Takahiro Fukudad, Toru Maekawad, Raymond L.D. Whitbya,b,
Sergey V. Mikhalovskya,b, Mohammed Alavijehc, Ananth S. Pannalaa,⁎, Dipak K. Sarkera
a
Biomaterials & Drug Delivery Research Group, School of Pharmacy & Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, United Kingdom
b
Department of Chemical Engineering, Faculty of Engineering, Nazarbayev University, 53 Kabanbay Batyr Avenue, Astana, Kazakhstan
c
Pharmidex, 14 Hanover Street, Mayfair, London W1S 1YH, United Kingdom
d
Bio-Nano Electronics Research Centre, Toyo University, Kawagoe, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: The development of liposome-nanoparticle colloid systems offers a versatile approach towards the manufacture
Nanoparticle of multifunctional therapeutic platforms. A strategy to encapsulate small metallic nanoparticles (< 4 nm) within
Liposome multilamellar vesicles, effected by exploiting electrostatic interactions was investigated. Two liposome-gold
Encapsulation nanoparticle (lipo-GNP) systems were prepared by the reverse-phase evaporation method employing cationic or
Electrostatic interaction
anionic surface functionalised particles in combination with oppositely charged lipid compositions with
Loading content
subsequent post-formulation PEGylation. Structural characterisation using electron microscopy and elemental
analysis revealed a regular distribution of GNPs between adjacent lipid bilayers of intact liposomes. Nanoparticle
encapsulation efficacy of the two lipo-GNP systems was revealed to be significantly different (p = 0.03),
evaluated by comparing the ratio of measured lipid to gold concentration (loading content) determined by a
colorimetric assay and atomic emission spectroscopy, respectively. It was concluded that the developed synthetic
strategy is an effective approach for the preparation of liposome-nanoparticle colloids with potential to control
the relative concentration of encapsulated particles to lipids by providing favourable electrostatic interactions.

1. Introduction
The design and application of functional nanosystems has become
well established in recent years encompassing fields of biology,
medicine, photonics, catalysis and materials science (Roco, 2004).
The driving force behind this development can be ascribed to a common
goal: to create new materials and devices with superior properties,
functions, efficiency and safety. As increasingly sensitive and complex
nanosystems are created the demand to find convenient approaches of
protecting and preserving the integrity of functional components has
arisen (Albet-Torres and Månsson, 2011). In addition, the ability to
isolate individual functional materials presents a new strategy of
controlling in situ responsive systems and potentially, self-assembling
materials.
A diverse array of nanomaterials are currently under investigation
and as a result an increase in human and environmental contact appears
inevitable with potentially hazardous consequences (Faraji and Wipf,
2009; Hristozov and Malsch, 2009). Metallic nanoparticles in particular
receive considerable attention, owing to their unique exploitable size
and shape-dependent physical and chemical properties (Link and El-
Sayed, 2000). Although frequently reported as intriguing candidates for
a range of applications, few options are available to avoid their
associated toxicity and side-effects without significantly altering surface
chemistry (Lewinski et al., 2008). Overcoming this, strategies of
incorporating metallic nanoparticles within traditional “soft matter”
materials such as polymers and lipids in a non-covalent manner are
being investigated (Puri et al., 2009). This approach is hypothesised to
combine advantageous features whilst enabling individual nanoparti-
cles to retain their native characteristics (Cattaneo et al., 2010;
Longmire et al., 2011). Moreover, combining multiple components into
nanosystems facilitates the implementation of multi-functionality, such
as in vivo targeting capabilities combined with controlled drug release
or responsive in situ activity (Mura et al., 2013; Muthu et al., 2014).
Sharing this notion, nanoparticles have been incorporated into
conventional “delivery vehicles” such as liposomes, which are artifi-
cially prepared closed vesicles that are composed of lamellae of lipid
bilayers surrounding an aqueous core (Chang et al., 2012). The
vesicular structure provides a non-associated surface passivation layer

⁎
Corresponding author at: School of Pharmacy & Biomolecular Sciences, University of Brighton, Lewes Road, Brighton BN2 4GJ, United Kingdom.
E-mail address: a.s.v.pannala@brighton.ac.uk (A.S. Pannala).

http://dx.doi.org/10.1016/j.ejps.2017.05.001
Received 18 January 2017; Received in revised form 23 March 2017; Accepted 2 May 2017
Available online 03 May 2017
0928-0987/ © 2017 Elsevier B.V. All rights reserved.
G.A. Dichello et al.
that protects, preserves and isolates the nanomaterials (Nam et al.,
2013), whilst also providing the framework in which multiple func-
tional or therapeutic components can be combined into a single system
(Okuda and Kidoaki, 2012). In addition, the liposomal exterior surface
provides a platform for further modification whereby predictable in situ
or in vivo behaviour can be attained (Nag and Awasthi, 2013). Two
general classes of nanoparticle-in-liposome configurations have pre-
viously been reported with particles distributed within lipid bilayers
(Park et al., 2006) or the aqueous core (Hong et al., 1983). However,
incorporating nanoparticles into bilayers requires particles with hydro-
phobic surfaces, whilst entrapment within the aqueous core relies on
spontaneous encapsulation resulting in few particles per liposome
(Chen et al., 2006).
In the current study, electrostatic interactions between negatively
charged lipids and positively charged GNPs (NCL-PCG) and vice versa
(PCL-NCG) were investigated as an alternative driving-force to facilitate
entrapment of nanoparticles within liposomes. This concept is based on
previous reports, such as lipofection systems, where ionic macromole-
cules have been encapsulated within liposomes containing oppositely
charged ionic lipid compositions (Balazs and Godbey, 2010). GNPs
were selected as a proof-of-concept to encapsulate within liposomes
primarily because of their inherent chemical robustness and availability
of established techniques for surface engineering. Physicochemical
characterisation and morphological assessment of the synthesised
lipo-GNP systems was investigated. Short-term (48 h) colloidal stability
was evaluated and quantification of internalised gold and lipid
concentration was also determined.
2. Materials and methods
2.1. Materials
Lyophilised lipids: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
(DPPC); 2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DPPG);
1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP); 1,2-dipalmi-
toyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene gly-
col)-5000] (DPPE-PEG5000); 200 nm porous (13 mm diameter) poly-
carbonate filter membranes and “Avanti mini-extruder system” were
purchased from Avanti Polar Lipids (Alabaster, Alabama, USA).
Platinum-blue (dye) was obtained from Nisshin EM Corporation
(Shinjuku, Tokyo, Japan). Hydrogen tetrachloroaurate, triphenylpho-
sphine (TPP), 2-dimethylaminoethanthiol (DMET), 2-mercaptoethane-
sulfonate (MES), tetraoctylammonium bromide (TOAB), Sodium bor-
ohydride, bromothymol blue, Sephadex-G50, phosphate buffered saline
(PBS) and all other reagents were purchased from Sigma-Aldrich
Chemical Company (Gillingham, Dorset, UK).
2.2. Synthesis of gold nanoparticles
GNPs bearing cationic or anionic ligands on their surfaces were
prepared according to a previously reported two-step approach.
Triphenylphosphine (TPP) stabilised GNPs were first prepared via a
modified Brust method (Weare et al., 2000) and subsequently utilised in
interfacial-ligand exchange reactions with DMET or method (Warner
et al., 2000).
TPP-GNPs were prepared by dissolving hydrogen tetrachloroaurate
(0.25 g, 0.64 mmol) and TOAB (0.4 g, 0.73 mmol) in nitrogen sparged
water/toluene (13 ml/17 ml) followed by addition of triphenylpho-
sphine (0.58 g, 2.21 mmol) under vigorous stirring. Freshly prepared
aqueous sodium borohydride (0.35 g, 9.4 mmol, dissolved in 10 ml)
was then added at once by injecting directly into the organic phase,
triggering a colour change to dark purple. The resulting mixture was
stirred for 3 h under a nitrogen atmosphere before the organic phase
was isolated and washed (× 3) with distilled water. The product was
further washed with a series of hexane and methanol:water (2:3) to
remove the phase-transfer catalyst and unreacted starting materials.
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European Journal of Pharmaceutical Sciences 105 (2017) 55–63
TPP-GNPs (20 mg) were resuspended in dichloromethane (4 ml)
and added to an aqueous solution containing either MES (20 mg,
0.12 mmol in 2 ml) or DMET (40 mg, 0.28 mmol in 3 ml). An argon
atmosphere was introduced and the biphasic mixtures were vigorously
stirred to ensure sufficient contact between the two phases. Complete
transfer of the coloured nanoparticles into the aqueous phase occurred
within 4 h at which point it was extracted. The nanoparticle suspen-
sions were then washed (×3) with dichloromethane and GNPs were
isolated by ultracentrifugation (200,000 × g). Recovered products were
then re-suspended in ethanol (20 ml) and precipitated upon addition of
hexane (20 ml) using centrifugation (4000 × g) to remove excess
ligands. Resultant GNP powders were then dried under vacuum over-
night before resuspending in deionised water.
2.3. Formulation of gold nanoparticle encapsulated liposomes
Two individual formulations were prepared via a three-step strategy
following previously published protocols with modifications. Initially,
crude lipo-GNPs were prepared by reverse-phase-evaporation method
(Szoka and Papahadjopoulos, 1978) before undergoing size-extrusion
(Olson et al., 1979) and subsequent post-formulation lipid-PEG mod-
ification (Awasthi et al., 2004). Liposome suspensions were then
separated from unencapsulated GNPs and excess reagents using size-
exclusion chromatography (Chen et al., 2006).
Lipid compositions DPPC:DPPG (molar ratio 6.1:0.7) and
DPPC:DPTAP (molar ratio 6.1:0.8) were prepared by combining
lyophilised lipid powders and dissolving in chloroform:methanol
(2:1), providing a 2.5 mg/ml total lipid content. Aqueous GNP suspen-
sions with a concentration of 0.5 mg/ml were then combined with the
appropriate lipid solutions (1:1 v/v) and subsequently sonicated to
afford a white/brown emulsion-like mixture. The biphasic suspension
was then transferred onto a rotary evaporator for gradual removal of
the organic phase. Complete removal of organic solvents was achieved
within 1 h and resultant crude liposome suspensions were diluted in
PBS (pH 7.4) to provide a total lipid content of 1 mg/ml. Aliquots (1 ml)
were then briefly sonicated before repeatedly (×15) passing through
200 nm porous polycarbonate membranes utilising an “Avanti mini
extruder system.” The temperature was maintained slightly above the
melting transition of the main lipid component, DPPC, (Tm = 41 °C)
and reduced to room temperature during the extrusion process.
Aliquots were then either set-aside for purification or added immedi-
ately to 0.2 ml portions of a preheated (50 °C) aqueous solution of
DPPE-PEG5000 (0.01% w/v) in glass vials. The temperature was
maintained for 30 min before allowing the liposomal suspensions to
naturally reach room temperature.
To separate liposomes from unencapsulated GNPs and excess
reagents, size-exclusion chromatography was performed using
Sephadex-G50 gel as the stationary phase and PBS (pH 7.4) as eluent.
Fractions were collected (1 ml) and the first five fractions identified to
contain lipids by spot staining with bromothymol blue (pH 6) in 10% v/
v aqueous ethanol solution, were combined and stored at room
temperature.
2.4. Hydrodynamic particle size analysis of liposomes
Lipo-GNPs hydrodynamic size distributions and dispersity values
were determined using a Zetasizer, (Malvern, NANO ZS90 series),
operating in DLS mode at 25 °C with measurements recorded at a 90-
degree detection angle. Representative samples from each batch
(n = 6) were diluted in PBS buffer (0.45 μm sterile filtered), gently
shaken and rested for 10 min before analysis.
2.5. Zeta potential analysis of liposomes
The same lipo-GNPs specimens prepared for size analysis were
studied using a Zetasizer, (Malvern, NANO ZS90 series), operating in
G.A. Dichello et al.
zeta mode at 25 °C using disposable folded capillary cells (DTS1070,
Malvern Instruments Ltd., UK). Measurements were also recorded for
liposome samples that had not undergone PEG modification.
2.6. Study of gold encapsulation using TEM
The morphology of liposomes and encapsulation of GNPs was
studied using a high-resolution transmission electron microscope
(TEM - JEOL JEM-2200) operating at an accelerating voltage of
200 kV. Samples were diluted 10-fold with distilled water and pipetted
onto hydrophilised, carbon-formvar coated 200-mesh copper grids
(Agar Scientific, UK) before wick drying excess solution with filter
paper. Negative staining of specimens was achieved by exposing grids
preloaded with sample to platinum-blue (dye) for 30 s prior to wick
drying; all specimen grids were allowed to rest for 30 min before
analysis. The same unstained-specimens were used for energy disper-
sive X-ray spectroscopy (EDS) operating in scanning-transmission
(STEM) mode, utilising an integrated JEOL-JED-2300 EDS detector.
2.7. Topographical assessment of lipo-GNPs using SEM
Lipo-GNPs surfaces were studied using a field-emission scanning
electron microscope (SEM - Zeiss – Sigma 300) operating at an
accelerating voltage of 5 kV using a secondary electron detector (Cho
et al., 2015). Samples were diluted 10-fold with distilled water and
10 μl was deposited onto silicon wafer mounted on aluminium speci-
men stubs. Specimens were snap-frozen by submerging in liquid
nitrogen and subsequently freeze-dried (Christ®-ALPHA 1–4) before
sputter coating (5 nm) with platinum (Quorum Q150T ES Turbo-
Pumped coater device).
2.8. Quantification of gold encapsulation using AES
Quantification of encapsulated gold was determined using an
Agilent 4100 microwave-plasma atomic emission spectrometer (AES)
according to a previously reported protocol with modifications
(Chithrani et al., 2006). TPP-GNPs were digested in aqua regia
(HCl:HNO3) for 24 h before serially diluting to prepare a range of
calibration standards (0–20 μg/ml). For the analysis of lipo-GNP
samples, 200 μl aliquots were mixed with chloroform (1 ml), vortexed
for 30 s and the two phases separated by centrifugation (300 × g,
5 min). The aqueous phase was then isolated, dried under mild heating
and residues were digested in aqua regia (0.2 ml) for 24 h. Specimens
were then accurately made up to a total volume of 2 ml with distilled
water. To attain fitting baselines, blank solutions were prepared by
digesting 0.1 mmol of the appropriate ligand (TPP, DMET or MES) in
aqua regia and diluting with distilled water. In validation, stock
solutions of DMET and MES functionalised GNPs (6.25 μg/ml) were
serially spiked (0, 2, 4, 6, 8 ml) with further digested GNP solutions
(25 μg/ml) in 25 ml volumetric flasks. Specimens were introduced to
the nebuliser at a pump rate of 80 rpm with a 15 s stabilisation time
before measurements were recorded (λ = 267.595 nm).
2.9. Quantification of DPPC lipid content in formulations
Quantification of the main lipid component (DPPC) was achieved by
Table 1
A summary of data obtained from dynamic light scattering and zeta potential analysis of lipo-GNPs after their preparation (T0), after 48 h of storage (T48), with and without PEGylation
(mean ± standard deviation, n = 6).
Initial DLS measurements (T0)
Size (d·nm) Dispersity
NCL-PCG 183 ± 12 0.08 ± 0.02
PCL-NCG 176 ± 8 0.13 ± 0.05
European Journal of Pharmaceutical Sciences 105 (2017) 55–63
application of the Stewart Assay (Stewart, 1980). An ammonium
ferrothiocyanate (AF) standard solution was prepared by dissolving
ferric chloride hexahydrate (27.03 g, 0.1 mol) and ammonium thiocya-
nate (30.4 g, 0.4 mol) in 1 l of distilled water. Lyophilised DPPC was
dissolved in chloroform and serially diluted to prepare a range of
calibration standards (0–80 μg/ml). Aliquots of each standard (1 ml)
were then combined with AF (2 ml) and vortexed before isolating the
organic phase by centrifugation (300 × g, 5 min). Prior to analysis, the
volume was corrected with chloroform (2 ml) to account for any
evaporation. For the analysis of lipo-GNP specimens, 100 μl of a sample
suspension was sonicated for 5 min before the addition of the AF
reagent. In validation, a stock solution containing DPPC (200 μg/ml),
DPPG (20 μg/ml) and DPTAP (20 μg/ml) was prepared in PBS buffer
and serially spiked (0, 1, 2, 3, 4 ml) with a solution of DPPC (200 μg/
ml) in 50 ml volumetric flasks before treating with AF reagent. The
absorbance of specimens and standards was measured using a Perki-
nElmer LAMBDA-25 ultraviolet-visible spectrometer (λ = 485 nm).
3. Results
3.1. Preparation of gold nanoparticles
The GNPs utilised in this investigation were prepared by a two-stage
wet synthesis where TPP-GNPs are first synthesised before undergoing
interfacial-exchange reactions with thiol-containing ionic ligands. The
first step ensures the formation of uniform, spherical GNPs of the
desired size, considered a critical parameter for encapsulation within
liposomes. The second stage introduces the desired surface ligands
whilst crucially retaining the original particle size (Warner et al., 2000).
Characterisation via TEM gives an average gold core size of
2.8 ± 1.2 nm for DMET-GNPs and 2.6 ± 1.2 nm for MES-GNPs
(n = 100), comparable to previous reports using this approach
(Warner et al., 2000). As expected, zeta potential analysis reveals GNPs
display a positive surface potential of + 15.1 ± 1.2 mV and a negative
surface potential of −17.5 ± 1.4 mV when functionalised with either
cationic DMET or anionic MES ligands, respectively. The utilised
approach was found to be a simple and highly reproducible method
affording sufficient yields and recovery of GNPs for the purpose of this
investigation.
3.2. Zeta potential and PEGylation of lipo-GNP formulations
To verify the successful insertion of PEG into the exterior lamella of
prepared lipo-GNPs, zeta potential measurements (Table 1) were
performed before and after post-formulation PEG modification for each
batch (n = 6). For NCL-PCG a reduction in magnitude was observed
from − 20.1 ± 0.2 mV to − 5.11 ± 0.59 mV, whilst PCL-NCG shifted
from a positive potential of + 2.9 ± 0.3 mV to negative
− 3.61 ± 0.32 mV. As revealed, a characteristic charge-shielding
effect takes place upon DPPE-PEG insertion, in agreement with previous
reports of liposomal modification with PEG (Nakamura et al., 2012).
The low magnitude negative surface potential displayed by both
systems can be ascribed to PEG itself (Nakamura et al., 2012).
DLS measurements after 48 h (T48) Zeta potential measurements (mV)
Size (d·nm) Dispersity Without PEGylation After PEGylation
148 ± 2 0.08 ± 0.02 − 20.1 ± 0.20 − 5.11 ± 0.59
147 ± 1 0.08 ± 0.05 + 2.94 ± 0.30 − 3.61 ± 0.32
57
G.A. Dichello et al.
3.3. Hydrodynamic size and colloidal stability of liposomes
Colloidal particle size data obtained for six individual batches
(n = 6) of NCL-PCG and PCL-NCG using dynamic light scattering is
summarised in Table 1 with values expressed as mean ± standard
deviation. Average hydrodynamic diameters of 183.0 ± 12.3 nm for
NCL-PCG and 175.9 ± 8.1 nm for PCL-NCG were recorded with
dispersity values of 0.08 ± 0.02 and 0.17 ± 0.06 respectively. Aver-
age liposome sizes below 200 nm are observed, reflecting the pore size
utilised in the extrusion process. Low dispersity values and size
variability for both systems suggest a high degree of colloidal homo-
geneity.
As an estimate of colloidal stability specimens used in size measure-
ments were reanalysed under identical conditions after 48 h. Values of
147.6 ± 2.3 nm for NCL-PCG and 147.4 ± 1.2 nm for PCL-NCG
reveal a reduction in hydrodynamic particle size for both systems.
Statistical comparison (paired two-tail student t-test) reveals the
decrease in particle size to be significant from initial measurements
for both NCL-PCG (p = 0.0014) and PCL-NCG (p = 0.0005). Dispersity
values (Table 1) remained comparably low for both systems (p > 0.5)
indicative of uniform colloidal particles, suggesting size reductions are
a universal trend.
3.4. Morphological assessment and elemental analysis of lipo-GNPs
SEM was utilised to study the morphological features of lipo-GNPs
evidencing whether GNPs were present on the exterior surfaces of
liposomes. TEM enabled visualisation of internalised nanoparticles as a
result of the electron density difference between gold and lipids.
Negative staining of specimens with an electron-dense dye was then
employed to reveal whether liposomal structures were intact. This was
evidenced by the internal liposomal structure remaining unstained
relative to the stained background (De Carlo and Harris, 2011). STEM-
EDS provided elemental maps and comprehensive spectra enabling
evaluation of gold distribution and identification of other elements
present. Micrographs of SEM, TEM and corresponding EDS for NCL-PCG
Fig. 1. Representative images of NCL-PCG (A): SEM micrograph (B): STEM micrograph of an individual vesicular structure with (C): corresponding EDS map for gold. (D, E): TEM
micrographs of negatively stained specimens.
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European Journal of Pharmaceutical Sciences 105 (2017) 55–63
and PCL-NCG are presented in Fig. 1 and Fig. 2 respectively. From SEM
micrographs (Fig. 1A and Fig. 2A), lipo-GNPs are revealed to display
uniform surface morphologies with smooth exteriors and no features
that can be ascribed to surface-exposed GNPs. Structures appear to be
well-dispersed, spherical entities consistent with stable colloidal parti-
cles. TEM micrographs of liposomes negatively stained with an
electron-dense platinum-based dye (Fig. 1B, C and Fig. 2B, C) reveal
intact, spherical, vesicular structures with well-defined boundaries even
when in close proximity to one another. Features characteristic of GNPs
are revealed within the confinements of these structures and appear as
densely contrasted regions relative to the unstained background. In
both systems, such features are observed in high numbers frequently
appearing concentrated among the bilayer region of vesicles. This is
further highlighted in Fig. 1B and Fig. 2B where repeating “rings” of
features ascribed to GNPs are observed. To confirm these observations,
representative structures were selected for further elemental analysis.
STEM micrographs and corresponding elemental maps of gold can be
seen in Fig. 1D, E and Fig. 2D, E with interpreted spectra provided in
Fig. 3. These observations confirm the previous assumptions, with
features appearing concentrated within liposomal structures ascribed to
GNPs correlating with the detection of gold in STEM micrographs and
EDS maps. Other elements revealed from spectra include carbon,
oxygen and phosphorus which can be assigned to the presence of
lipids, sulphur which is indicative of ligands on GNP surfaces and
copper from TEM specimen grids used in the analysis.
3.5. Quantifying liposomal gold encapsulation
The concentration of internalised gold was determined by atomic
emission spectroscopy using a protocol previously applied to quantify
GNP uptake in cellular studies (Chithrani et al., 2006). A calibration
plot was prepared between 0 and 20.0 μg/ml of digested TPP-GNPs
(Fig. 4A) and the equation of least squares regression line was derived
(y = 8507.5x + 204.02, R2 = 0.998). Corresponding concentrations of
gold in lipo-GNPs specimens (n = 6) were then calculated with NCL-
PCG revealed to contain 14.9 ± 6.1 μg/ml and PCL-NCG containing
G.A. Dichello et al.
Fig. 2. Representative images of PCL-NCG (A): SEM micrograph (B): STEM micrograph of an individual vesicular structure with (C): corresponding EDS map for gold. (D, E): TEM
micrographs of negatively stained specimens.
Fig. 3. Interpreted EDS spectra corresponding to STEM images displayed in Figs. 3B and 4B (A): NCL-PCG and (B): PCL-NCG.
6.2 ± 2.9 μg/ml. The procedure was validated using the method of
standard additions in a representative matrix, recovery (n = 3) of
DMET-GNPs was calculated to be 93.6 ± 4.6% and MES-GNPs
98.8 ± 7.4% with statistical comparison (unpaired student t-test)
revealing no significant difference (p > 0.05) between recoveries
(Fig. 4B).
3.6. Quantifying DPPC lipid content in the lipo-GNP formulations
Measurements of DPPC lipid concentration were determined by the
colorimetric Stewart assay, a well-established technique that is suitable
for the detection of sub-micromolar concentrations of phosphatidylcho-
line lipids in the presence of phosphate buffers utilised in this
investigation (Stewart, 1980). A calibration plot ranging from 0 to
40.0 μg/ml of DPPC lipid was prepared (Fig. 5A) and the equation of
least squares regression line was derived (y = 0.0103x + 0.0291,
R2 = 0.992). Corresponding concentrations of DPPC in lipo-GNP speci-
mens (n = 6) were then calculated with NCL-PCG revealed to contain
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European Journal of Pharmaceutical Sciences 105 (2017) 55–63
202.4 ± 39.7 μg/ml and PCL-NCG 148.0 ± 30.4 μg/ml. The proce-
dure was validated using the method of standard additions in a
representative matrix, recovery of DPPC was calculated to be
103.7 ± 7.3%, (red marker, Fig. 5B).
4. Discussion
Two distinct lipo-GNP systems were formulated by exploiting
electrostatic interactions between GNP surfaces and lipids that consti-
tute the liposomes. This interaction between opposing charges of the
two components facilitates their amalgamation as the lipids self-
organise to form vesicular structures. The proposed configuration
(Fig. 6) shows a regular nanoparticle distribution within multilamellar
liposomes located at the peripheral region of the central aqueous core
and at the interface of adjacent lipid bilayers and separating layers of
water (Shaheen et al., 2006).
Adopting the reverse-phase evaporation method, interactions be-
tween lipid “head groups” and GNPs at the organic-aqueous interface
G.A. Dichello et al. European Journal of Pharmaceutical Sciences 105 (2017) 55–63

Fig. 4. Quantification of gold encapsulation by AES (A): Calibration plot of digested TPP-GNPs with (insert) highlighting lower concentrations. (B): Bar graph comparing the relative (%)
recovery of DMET-GNPs and MES-GNPs. Values are reported as mean with error bars representing standard deviation. C): Concentration of gold determined for NCL-PCG and PCL-NCG
(mean ± standard deviation, n = 6).

Fig. 5. Quantification of DPPC lipid using the Stewart Assay (A): Calibration plot for DPPC lipid (B): plot of standard additions with extrapolated value (red marker) corresponding to
recovery of DPPC lipid in a representative sample matrix (C): Concentration of DPPC determined for NCL-PCG and PCL-NCG (mean ± standard deviation, n = 6). (For interpretation of
the references to colour in this figure, the reader is referred to the web version of this article.)

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Fig. 6. Proposed structure of lipo-GNPs with the underpinning lipid-nanoparticle interaction at the aqueous-bilayer interface highlighted (insert). Lipid structures utilised in the
preparation of NCL-PCG and PCL-NCG are presented (A): DPPC, (B): DPTAP, (C): DPPG.

Fig. 7. Simplified representation of the processes involved in the preparation of lipo-GNPs. (A): Lipid molecules interacting with GNPs (white arrow) at the aqueous-organic interface (B):
GNP-liposome formation, (C): size extrusion, (D): post-formulation PEG insertion (E): PEG-grafted GNP-liposomes after size exclusion chromatography.

bring the two components into close proximity (white arrows, (Fig. 7A,
B). As the organic solvent is depleted lipid molecules transfer into the
aqueous phase and self-organise to form closed lipid-bilayers (Shailesh
et al., 2009). Associated GNPs subsequently become entrapped
throughout the multilamellar vesicular structures. Crude lipo-GNPs
are then resized via size extrusion, where liposomes are repeatedly
forced through 200 nm porous filter membranes. Starting extrusion at
an elevated temperature and working downward facilitated the process
and ensured that agglomerates were not formed before the next stage.
The resultant suspensions were then immediately subjected to post-
formulation PEGylation using a PEG grafted lipid (DPPE-PEG-5000).
This was performed whilst passing through the melting-transition
temperature (Tm) of the main lipid component (DPPC, 41 °C), which
supports insertion into the outermost lamella of liposomes. Removal of
excess GNPs and reagents was then achieved by size-exclusion gel
chromatography, where the larger particles (i.e. liposomes) elute from
the column quicker than the smaller particles and molecules (i.e. excess
nanoparticles and unincorporated PEG) as they pass through a porous
matrix (Bozzuto and Molinari, 2015). Although visually a band of
nanoparticles could be seen retained within the chromatography
column, as a precautionary measure recovery of liposomes was
restricted to a total of five 1 ml aliquots. This step was taken to ensure
that subsequent characterisation and quantification was representative
of lipo-GNPs and encapsulated gold exclusively.
Microscopy and elemental analysis suggests that the strategy
employed is a promising route for the preparation of liposomes with

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internalised small (< 4 nm) metallic nanoparticles. Although compar-
able strategies exploiting electrostatic attractions have previously been
utilised to encapsulate a range of materials within liposomes, according
to available literature this approach has not been applied for the
internalisation of metal nanoparticles. Furthermore, as a result of
electrostatic interactions between lipid “head groups” and particle
surfaces, GNPs appear regularly distributed at the liposomal bilayer-
aqueous interface. This leads to the proposal that nanoparticle arrange-
ment is dissimilar from previous reports of either encapsulation of
hydrophobic nanoparticles within bilayers or hydrophilic nanoparticles
within the aqueous core of liposomes. The concept of using electrostatic
interactions has previously been demonstrated for entrapment of
enzymes and DNA within liposomes (Colletier et al., 2002; Ma et al.,
2007).
Surface charge of cationic DMET-GNPs and anionic MES-GNPs as
determined by measuring the zeta potentials were initially revealed to
be of a comparable magnitude. After subsequent encapsulation within
liposomes consisting of lipid compositions at identical mole ratios of
zwitterionic (DPPC) and opposing monoionic lipids (DPTAP or DPPG),
zeta potentials of a dissimilar magnitude were observed (Table 1). The
parameters that were considered to be potentially contributing towards
the observations in this study include the quantity of GNPs associated
per liposome (Mady et al., 2012), ligand density on GNP surfaces
(Laaksonen et al., 2006) and charge distribution of the dissimilar lipid
structures (Langner and Kubica, 1999). Consequently, after PEGylation,
both systems again display comparable zeta potential values as a result
of characteristic charge-shielding effects of PEG. This phenomenon
arises as a result of sheltering the potential of the electrical double-
layer, which in turn effectively diminishes zeta potential (MacLachlan,
2007). These results imply that although NCL-PCG and PCL-NCG
appear comparable at the surface, their internal properties are different.
Further investigation is required to probe the “layer by layer” or sub-
surface properties beneath the outer PEG stratum and adjacent lamella
to determine how these characteristics vary and what if any implica-
tions that this may entail. Introducing PEG to the surface of prepared
lipo-GNPs follows recent trends in literature that stem from a catalogue
of known advantageous biological and technological properties
(Alcantar et al., 2000; Nag and Awasthi, 2013). Incorporating PEG
exclusively into the exterior lamellar of liposomes using a post-
formulation approach avoids reducing the internal aqueous volume
(Szoka and Papahadjopoulos, 1978), which could potentially hinder
encapsulation of GNPs. In addition, post-formulation modification
avoids the use of excessive quantities of lipopolymers such as PEG that
can make size-extrusion processing difficult (Nakamura et al., 2012).
Particles in suspension must remain dispersed at equilibrium to
avoid common instability phenomena such as aggregation or phase
separation, which is often achieved by electrostatic repulsion or steric
stabilisation. In this investigation steric hindrance was successfully
implemented by PEGylation of lipo-GNP surfaces with the PEG-grafted
lipid DPPE-PEG5000. Previous reports of using PEG with an average
molecular weight of ~5000 g/mol was reported to provide liposomes
with a 5 nm thick “coating” that acts as a barrier, shielding the surface
from the external environment (Woodle et al., 1994). This approach
was taken in an attempt to prevent potential inter-particle interactions
between exterior-surface bound GNPs and neighbouring liposomes. To
confirm this, lipo-GNP hydrodynamic sizes were monitored upon
storage, a common approach enabling early identification of instability
(Mengual et al., 1999). Liposomal suspensions retained a high degree of
uniformity and narrow size distributions after 48 h (Table 1) with no
aggregate formation or phase separation observed. Both liposomal
systems however did display statistically significant decreases in
particle sizes, although dispersity values, which provide a measure of
colloidal homogeneity, remained consistently low indicating this to be a
universal trend. These observations agree with previously reported
findings where interactions between phospholipids and ionic nanopar-
ticles on the exterior surface of liposomes were responsible for lipid
62
European Journal of Pharmaceutical Sciences 105 (2017) 55–63
reorganisation and a decrease in particle size (Wang et al., 2008). This
was suggested to occur as a consequence of alteration to the tilt-angle of
lipid head groups, resulting in an increase in lipid-tail density and
tighter packing, typical of solid-phase bilayer behaviour (Somerharju
et al., 1999). Further investigation is warranted to determine whether
the mechanism of particle size reduction observed in this investigation
can be explained by a similar mechanism and whether such properties
could be advantageous.
Currently the evaluation of nanoparticle encapsulation within
liposomes primarily involves qualitative and visual elucidation alone,
which can be subjective (Mady et al., 2012). Even in instances
investigating how the concentration of nanoparticles in liposomal
systems influence resultant performance appears to provide no direct
quantification in support (Adhikari et al., 2015). As a matter of safety, it
is vital that nanosystems for therapeutic applications are prepared with
absolute precision. Further, as highlighted above, the encapsulation and
distribution of nanoparticles in liposomes can determine their resultant
physical and chemical properties making the gold to lipid ratio a critical
design parameter. Although the influence of this ratio on liposomal
bilayer properties has not been investigated in this study, previous
reports have demonstrated a range of effects. Among which the
formation of nano-pores within lipid bilayers that is dependent on lipid
composition, nanoparticle functionality and concentration and ensuing
electrostatic interactions (Lu et al., 2015), which may or may not be
desirable for specific applications. In an attempt to standardise the
evaluation of nanoparticle encapsulation and facilitate the development
of analogous systems, the concentration of gold and the main lipid
component DPPC were determined in parallel on the same samples.
Corresponding loading content (%) was calculated to reveal the ratio of
gold to lipid, providing a quantitative metric that can be compared to
assess encapsulation efficacy. This criterion has previously been applied
to evaluate the encapsulation of drugs (Xing et al., 2004) and was used
here to highlight the relationship between lipid compositions and GNPs
of different functionality. From the ratio of encapsulated gold to DPPC
lipid measured (n = 6), loading content (%) values of 7.3 ± 2.6% for
NCL-PCG and 3.1 ± 1.5% for PCL-NCG were calculated (mean ±
standard deviation). Statistical comparison (unpaired student t-test)
revealed the ratio of gold to lipid to be significantly different for the two
systems investigated (p = 0.03). Considering the two developed lipo-
GNP systems differ only by inclusion of monoionic lipid (DPPG or
DPTAP) and GNP functionality. It is hypothesised that ensuing variable
electrostatic interactions between the lipids and gold facilitates the
entrapment process and is responsible for the differences in observed
encapsulation efficacy. Parameters considered likely to be contributing
towards this electrostatic process include the ligand density on GNP
surfaces and the relative charge distribution of both the lipid and ligand
molecules (Laaksonen et al., 2006; Langner and Kubica, 1999).
Supporting these findings, electrostatic attractions have previously
been recognised as an essential mechanism for the encapsulation of
macromolecules in liposomes (Colletier et al., 2002), including lipofec-
tion systems on which the current synthetic strategy was based (Ma
et al., 2007). Further studies have highlighted that electrostatic
attraction between lipids and macromolecules are the primary factor
determining the encapsulation potential (Hwang et al., 2012), with
experimental conditions that diminish the interaction significantly
reducing efficacy (Shariat et al., 2014). Additional parameters identi-
fied from these studies that have not been investigated in the current
study but could be examined in future to provide optimal conditions to
increase the loading content of analogous GNP-lipo systems include
buffer composition, pH, ionic strength and liposomal size (Hwang et al.,
2012). On this premise, it is suggested that by selecting GNPs and lipids
to provide favourable electrostatic interactions, the reported approach
could be used to obtain the desired quantity of nanoparticles per
liposome.
G.A. Dichello et al.
5. Conclusion
In summary, a novel strategy of encapsulating GNPs within multi-
lamellar liposomes has been developed exploiting electrostatic interac-
tions between ionic lipid compositions and particle surfaces. Evaluation
of lipo-GNP morphology reveals particle distribution at the bilayer-
aqueous interface, differing from previous reports of nanoparticles in
bilayers or within the aqueous core of liposomes. The prepared
colloidal particles remain as dispersed and uniform populations for at
least 48 h but appear to experience a universal reduction in particle
size. Determining the ratio of gold to hydrated lipid provides a simple
approach to assess encapsulation efficacy demonstrating standardised
evaluation. Using a combination of variable lipid compositions and
GNP functionalities appears to influence the encapsulation process and
could be exploited for further control and optimisation. The reported
strategy of utilising electrostatic interactions for the encapsulation of
metallic nanoparticles is envisaged as a platform for the development
and study of analogous nanosystems.
Acknowledgments
Funding support was provided by the Medical Research Council
(MR/J006661/1) as part of an Industrial CASE studentship award in
collaboration with Pharmidex Pharmaceutical Services. Authors would
like to further thank associates from the Bio-Nano Electronics Research
Centre (Japan), for access and support during electron microscopy
studies.
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