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Article
Journal of
Nanoscience and Nanotechnology
Vol. 16, 1–6, 2016
www.aspbs.com/jnn
Ghee Butter as a Therapeutic Delivery System
Kishore Balasubramanian1†, Michael Evangelopoulos1†, Brandon S. Brown1, Alessandro Parodi1,
Christian Celia12,ImanK.Yazdi
13, and Ennio Tasciotti1∗
1Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA
2Department of Pharmacy, University of Chieti – Pescara “G. d’Annunzio”, Chieti, 66100, Italy
3Department of Biomedical Engineering, University of Houston, Houston, TX 77204, USA
Solid lipid nanoparticles carrying a chemotherapeutic payload (i.e., temozolomide, TMZ) were syn-
thesized using ghee, a clarified butter commonly used in traditional medicine and food products.
Ghee solid lipid nanoparticles (GSLN) were characterized through dynamic light scattering, scan-
ning electron microscopy, and UV-visible spectrometry. Formulations were generated with varying
ratios of surfactant to lipid, resulting in a maximum TMZ entrapment efficiency of ∼70%. Opti-
mal formulations were found to have an average size and polydispersity of ∼220 nm and 0.340,
respectively. Release kinetics revealed TMZ-loaded GSLN (TMZ@GSLN) retained 10% of its pay-
load at 2 h with ∼53% released in 5 h. Metabolic activity on human umbilical vein endothelial cells
(HUVEC) revealed GSLN treatment resulted in an increase in viability following 3 d while treatment
of glioblastoma LN-229 cells with TMZ@GSLN resulted in a significant decrease. Evaluation of dif-
fusion of TMZ across a reconstructed HUVEC monolayer demonstrated TMZ@GSLN resulted in a
significantly higher diffusion of drug when compared to free TMZ. This data suggests GSLN pose a
promising delivery vehicle for TMZ-based therapeutics. Collectively, this data demonstrates GSLN
exhibit favorable drug carrier properties with anti-proliferative properties in glioblastoma cancer cells.
Keywords: Ghee, Nanoparticles, Drug Delivery, Lipid.
1. INTRODUCTION
Systemic administration of therapeutics often requires
large doses to compensate for first pass metabolism,1
drug resistance,2and dynamic changes to the tumor
microenvironment.3Recently, drug delivery systems
have been proposed as promising alternatives to con-
ventional treatments due to unique features such as
biodegradability4–7 and the ability to combine therapeutics
with diagnostic features.8–11 Furthermore, encapsulation of
free drug into a drug delivery vector provides several
advantages: (i) decreased liver metabolism;12 (ii) improved
delivery13 and pharmacokinetics;14 (iii) selective targeting
through surface modifications.15–17
Solid Lipid Nanoparticles (SLN) have been explored
as alternatives to traditional colloidal nanocarriers due
to the beneficial release kinetics of encapsulated com-
pounds and enhanced stability of chemically-sensitive
lipophilic ingredients.18 SLNhavebeenshowntorange
from 50 to 1000 nm in size and are typically developed
∗Author to whom correspondence should be addressed.
†These two authors contributed equally to this work.
by dispersing biocompatible lipids in aqueous surfac-
tant solutions (e.g., Tween 80).19 Furthermore, SLN have
been shown to increase the efficacy of several therapeutic
agents,2021 making them favorable drug delivery systems.
This, combined with unique properties such as small size,
large surface area, high drug loading, biocompatibility, and
low toxicity have sparked the creation of various SLN-
based formulations.22–25
In this study, we developed SLN using ghee, a clari-
fied butter commonly used in Indian and Pakistani cook-
ing. Ghee has played an integral role in Ayurvedic
medicine (i.e., Hindu traditional medicine) as a homeo-
pathic treatment.26 Although, traditionally, ghee has been
used as a primitive transport vector for various herbal
preparations,27 more elaborate studies have demonstrated
ghee as an antioxidant with antiatherogenic potential.2829
This, together with ghee’s downregulation of enzymatic
activity associated with carcinogen metabolism30 led us
to investigate ghee’s potential as a chemotherapeutic drug
delivery system. Composed primarily of myristic, palmitic,
and searic fatty acids,3132 ghee consists of all the neces-
sary components for SLN production.33
J. Nanosci. Nanotechnol. 2016, Vol. 16, No. xx 1533-4880/2016/16/001/006 doi:10.1166/jnn.2016.12623 1
Ghee Butter as a Therapeutic Delivery System Balasubramanian et al.
Herein, our group investigated the physiochemical
properties and therapeutic potential of ghee-based SLN
(GSLN). Firstly, we evaluated the optimal parameters for
temozolomide (TMZ) entrapment, an oral chemotherapeu-
tic used to treat brain and skin cancers,34 by varying the
lipid to surfactant ratios. Next, GSLN were evaluated for
their ability to deliver and release a payload across a
reconstructed endothelial monolayer to glioblastoma cells.
Finally, cell viability was assessed on both endothelial and
glioblastoma cancer cells, showcasing GSLN as a promis-
ing drug delivery vector for TMZ transport.
2. MATERIALS AND METHODS
2.1. Materials
HPLC grade TMZ (>99% purity AK Scientific, Cata-
log ID #H027) was purchased from AK Scientific (AK
Scientific, Inc., Union City, CA). Ghee was obtained
from Pure Indian Foods Inc. (Princeton Jct., New Jersey).
All other chemicals were purchased from Sigma-Aldrich
(St. Louis, MO). Human Umbilical Vein Endothelial Cells
(HUVEC) were purchased from ATCC (American Type
Culture Collection, Manassas, VA). HUVEC were grown
in Endothelial Basal Media-2 and supplemented with
EGM-2 SingleQuots (Lonza). LN-229 cells were obtained
as a kind gift from M.D. Anderson Cancer Center and
were grown in F12/DMEM media supplemented with
10% (v/v) FBS and 1% (v/v) L-glutamine. All cells were
incubated and grown in a humidified atmosphere of 5%
CO2at 37 C.
2.2. GSLN Synthesis
The synthesis of GSLN was conducted using the
microemulsion technique.35 Briefly, the lipid phase (i.e.,
ghee) was heated until melted (30 C) and dissolved in
acetone. Next, 4 mg of TMZ was dissolved into 0.1 M
hydrochloric acid. The two solutions were sonicated and
added to a solution of 25 ml of water containing Polysor-
bate 80. The solution was stirred at room temperature for
15 min at 2,000 RPM and then stirred at 4 Cfor2hto
solidify the nanoparticles. It is well known that the entrap-
ment efficiency of SLNs is largely affected by the amount
of surfactant and lipids present in the formulation. In order
to determine the ideal combination of lipid and surfactant,
nine formulations of GSLN were synthesized and their
entrapment efficiencies were determined (See Table I for
formulations). The formulation with the highest entrap-
ment efficiency with minimal polydispersity would be used
in further studies.
Characterization of nanoparticles was performed by sus-
pending TMZ@ GSLN into 0.01 M phosphate buffer and
analyzed using a Zetasizer Nano ZA (Malvern) as previ-
ously reported.36 SEM was prepared for GSLN by diluting
in deionized water and placing on an aluminum stub. Sam-
ple was allowed to dry under a vacuum at room tempera-
ture and coated with palladium (∼7 nm thickness). Images
Tab l e I . Parameters used for the fabrication of GSLN formulations.
Sample name Polysorbate 80 (%) Lipid content (mg)
Formulation 1 0.8 50
Formulation 2 1.6
Formulation 3 2.4
Formulation 4 0.8 100
Formulation 5 1.6
Formulation 6 2.4
Formulation 7 0.8 150
Formulation 8 1.6
Formulation 9 2.4
were collected using the Nova NanoSEM 230 scanning
electron microscope.
2.3. Entrapment Efficiency
To determine the amount of drug in a given sample a TMZ
standard curve was constructed. Briefly, a stock solution
waspreparedbydissolving20mgofTMZin1mlof
DMSO. Serial dilution of the stock sample was conducted
to produce 20 samples each with a drug concentration of
half of the previous solution. Samples were analyzed using
a UV-visible spectrophotometer at 350 nm and absorbance
values were recorded. A standard curve was generated for
TMZ concentrations and the standard calibration was con-
ducted with a linear regression of y=21.514x.
The entrapment efficiency was reported by separating
the free drug through ultracentrifugation. Briefly, TMZ-
loaded GSLN suspensions were centrifuged at 40,000 rpm
for 4 hours at 4 C. Pellets from each of the samples were
isolated and the supernatants were stored. Supernatant
were analyzed using UV-Spectrophotometer at 350 nm and
absorbance values were recorded. The TMZ standard curve
was used to determine the amount of drug present in the
supernatant (unloaded drug). The entrapment efficiencies
for each formulation were determined and recorded using
the following formula.
EE %=DrugT
DrugSDrugT×100
where DrugTis the total amount of drug (4 mg) added to
GSLN during the preparation procedure and DrugSis the
amount of the drug in the supernatant.
2.4. Drug Release Study
Release of drug from TMZ-loaded GSLN was evaluated
by immersing TMZ-loaded GSLN into phosphate buffered
saline and placing the solution into dialysis bags with
a 100 kDa cut-off. Briefly, samples were subjected to
200 RPM rotation at 37 C. Samples were collected at
indicated time points and read using spectrophotometer.
Removed solution was replaced with an equal part of PBS.
2.5. Cell Viability and Proliferation
Effect of GSLN on HUVEC was performed by seed-
ing HUVEC into a 96 well plate at a concentration of
2J. Nanosci. Nanotechnol. 16, 1–6,2016
Balasubramanian et al. Ghee Butter as a Therapeutic Delivery System
9,700 cells cm−2. Cells were allowed to adhere overnight
and treated with GSLN. An MTT cell proliferation assay
was performed three days following treatment using man-
ufacturer suggested protocol. Briefly, 5 mg ml−1of MTT
was added to cells and incubated for 2 h at 37 C. Next,
MTT solution was aspirated and solvent (i.e., isopropyl
alcohol) was added and agitated on an orbital shaker for
30 min. Following agitation, samples were read at 540 nm
with a spectrophotometer.
LN229 glioblastoma cells were prepared by seeding
1,700 cells cm−2into a 96 well plate. Cells were incu-
bated overnight and treated with GSLN or TMZ@GSLN
for indicated time points. Following treatment times, MTT
assay was performed as previously described.
2.6. Vascular Barrier Permeation
Twelve vascular blood barrier models were prepared
using a HUVEC monolayer. Cell culture inserts were
seeded with 80,000 cells and allowed to adhere overnight.
Transendothelial resistance following treatment with
GSLN was determined using an EVOM2 Epithelial Volt-
meter (World Precision Instruments) in combination with
TX2 chopstick electrodes; measurements of resistance
were performed by placing the long end of the electrode
into the outer chamber and the short end of the electrode
into the inner chamber. To measure perfused drug, media
in the upper chamber was aspirated and replaced with
media containing GSLN. Following 1 h incubation, the
solution in the lower chamber was collected and analyzed
with a UV-Vis spectrophotometer.
2.7. Statistical Significance
All results were collected and analyzed in triplicate and
represented as a mean ±SD unless otherwise stated. Sta-
tistical significance was acquired using GraphPad Prism
6 software. For HUVEC cell viability and total drug per-
fusion, a Student t-test was employed. Cell viability of
glioblastoma cells were compared using a two-way anal-
ysis of variance followed by a Bonferroni post-test. Non-
linear regression analysis was presented using a one-phase
association fit with constraint Y0 set at 0.0 and a plateau
equal to 100. In all cases, asterisks denote the following
criteria: ∗for pvalues between 0.01 and 0.05, ∗∗for values
between 0.001 and 0.01, ∗∗∗for values between 0.001 and
0.0001, and ∗∗∗∗for values below 0.0001.
3. RESULTS AND DISCUSSION
3.1. Optimization of GSLN Fabrication
GSLN were fabricated following previously established
microemulsion procedures.35 Briefly, ghee was dissolved
in a solvent and mixed with suspended TMZ. The mix-
ture was then added to a solution containing surfactant and
allowed to mix. Following centrifugation, particles were
cooled and allowed to solidify. The size, polydispersity
index (PDI), and entrapment efficiency of GSLN is largely
affected by the amount of surfactant and lipids present
in the formulation. We synthesized nine distinct formu-
lations of GSLN using a total of 4 mg TMZ to deter-
mine the impact the ratio of lipids and surfactant have
on loading parameters. Each formulation was created with
either 0.8, 1.6, or 2.4% polysorbate 80 using either 50,
100, or 150 mg of ghee lipids (Table I). Physiochem-
ical properties of each formulation were then analyzed
through DLS and spectrophotometry (Figs. 1(a, b)). We
found that the particle size decreased as surfactant per-
centage (i.e., 0.8–2.4% Polysorbate) increased, however,
an increase in lipid content resulted in a linear increase
of particle size (Figs. 1(a), bars). Conversely, PDI was
observed to possess a decrease in polydispersity as surfac-
tant percentage increased (Fig. 1(a), black line). More so,
an increase in lipid content resulted in less monodisperse
particles. This can likely be attributed to the high surfac-
tant content reducing interfacial tension, allowing smaller
particles to become more homogenized in an aqueous
phase.37
Interestingly, a comparison of the PDI (Fig. 1(b), bars)
to entrapment efficiency (Fig. 1(b), black line) demon-
strated a decrease in entrapment efficiency for low lipid
content particles with a decrease in entrapment as sur-
factant percentage increased. Conversely, particles fabri-
cated with a 100 and 150 mg lipid content were observed
to exhibit an increase in entrapment as surfactant content
increased and, similarly, an increase in entrapment as lipid
content increased. Specifically, comparing particles fabri-
cated with 2.4% polysorbate 80, entrapment efficiency was
observed as 31, 62, and 70% entrapment of drug or 1.24,
2.48, and 2.8 mg of TMZ, respectively. This can likely be
Figure 1. DLS analysis of nine formulations of TMZ@GSLN devel-
oped with varying ratios of lipid to surfactant. A comparison of size
to PDI and PDI to entrapment efficiency demonstrates. Formulation
selected for all experiments indicated by red arrow. (C) SEM low- and
high-resolution (D) micrographs of GSLN following fabrication, (scale:
500 nm and 100 nm, respectively). The data is plotted as average.
J. Nanosci. Nanotechnol. 16, 1–6, 2016 3
Ghee Butter as a Therapeutic Delivery System Balasubramanian et al.
attributed to the higher lipid content preventing the leakage
of drug as previously reported in literature [38]. To maxi-
mize the therapeutic potential of TMZ, all future formula-
tion were created using 100 mg lipid and 2.4% Polysorbate
80 as indicated by red arrows. This formulation was cho-
sen over 150 mg formulations due to a more favorable
PDI. Scanning electron microscopy show low- (Fig. 1(c))
and high-resolution (Fig. 1(d)) images depicted spherical
GLSN.
3.2. Drug Loading and Release
To assess the therapeutic potential of TMZ@GSLN, an
evaluation of the TMZ loading and release was performed
on the formulation with the most favorable encapsulation
potential as described above (i.e., Formulation #6). First,
total TMZ entrapped in GSLN was evaluated with for-
mulations fabricated using 100 mg of lipid and found to
maintain a range of 1.7–2.5 mg of TMZ loaded or 41–62%
(Fig. 2(a)). Selecting the formulation with the highest
entrapment efficiency, the controlled release of TMZ from
GSLN was assessed in vitro over a 24 h time period using
dialysis membranes with a 100 kDa cutoff suspended in
physiologic buffer at 37 C under slight agitation. Follow-
ing 1 h of mixing, 8% of the drug was released while
54% of the drug was release following 5 h. After 22 h the
drug release leveled off at 80% (Fig. 2(b)). This delayed
release can be attributed to the TMZ being homogenously
dispersed in the lipid matrix as reported by other groups.39
Furthermore, applying a one-phase association fit curve
revealed a half time between 5.3 and 9.0 h demonstrating
GSLN are capable of carrying TMZ comparable to other
nanocarriers.4041
3.3. Cytotoxicity of TMZ-Loaded GSLN
We next evaluated the effects of GSLN on endothelial and
glioblastoma cancer cells in vitro. HUVEC were grown
to a confluent monolayer and continuously treated with
GSLN for three days (Fig. 3(a)). Following incubation
Figure 2. (A) Total amount of TMZ loaded into GSLN formulations
prepared with 100 mg lipid. Black arrow indicates formulation that was
selected for subsequent experiments. (B) Drug released from GSLN in a
physiologic buffer (pH 7.2) measured over 24 h. Trend line was deter-
mined using a one-phase associate fit curve.
Figure 3. (A) MTT assay was used to compare the effect of GSLN
on a HUVEC 3 d following treatment. ∗P<005. (B) Cellular viability
of glioblastoma cells compared to untreated and TMZ@GSLN treated
cells over 168 h. Data was normalized to GSLN-treated cells. ∗∗P<001,
∗∗∗P<0001, ∗∗∗∗P<00001.
with GSLN, cells viability was determined with an MTT
assay. Our results exhibit following incubation, cells
treated with GSLN exhibited a statistically significant
increase in cellular viability. Specifically, a 13% increase
in viability was observed for GSLN-treated cells demon-
strating the biocompatibility of GSLN. This observation is
likely attributed to the high quantity of conjugated linoleic
acid that has been previously reported to provide prolifer-
ative properties to other cells.42
Conversely, the treatment of glioblastoma LN-229 cells
with GSLN and TMZ@GSLN demonstrated that the
combination of GSLN and chemotherapy resulted in a
cumulative inhibition of glioblastoma metabolic activity
(Fig. 3(b)). Following the metabolic activity over the
course of one week, it was observed at 24 h, TMZ@GSLN
resulted in a 23% decrease in cell viability. At 48 h, a 28%
decrease was observed compared to GSLN-treated cells
while untreated cells displayed a 29% increase in cell
viability. Similarly, TMZ@GSLN-treated cells exhibited a
statistically significant decrease (58%, p<00001) in cell
viability at 168 h (i.e., one week) while untreated cells
demonstrated an exponential increase in proliferation. This
effect can likely be attributed to ghee’s anti-proliferative
properties for cancer cells due to the high level of con-
jugated linoleic acid. Previous results have demonstrated
in the presence of cancerous cells, high levels of linoleic
acid can inhibit the proliferation of breast and colon cancer
cells.43
3.4. Diffusion Across a Reconstructed
Endothelial Monolayer
Evaluation of the GSLN potential to favor the diffu-
sion of TMZ across a reconstructed HUVEC monolayer
was measured by analyzing the amount of drug deliv-
ered into the lower chamber of a cell culture insert as
depicted in Figure 4(a). In brief, an endothelium bar-
rier was constructed by seeding HUVEC onto the upper
chamber of cell culture inserts and allowed to adhere
overnight. To confirm the establishment of an endothe-
lial monolayer on the cell culture insert, scanning electron
4J. Nanosci. Nanotechnol. 16, 1–6,2016
Balasubramanian et al. Ghee Butter as a Therapeutic Delivery System
Figure 4. (A) Cartoon schematic of cell culture insert with HUVEC
monolayer (blue) in upper chamber (B) SEM micrograph of HUVEC
monolayer following 24 h incubation on the upper side of the cell culture
insert, (scale, 30 m). (C) TEER measurement of the HUVEC monolayer
following a 60 min treatment with GSLN. (D) Total drug diffused into the
lower chamber of the well following a 60 min. treatment. ∗∗∗∗P<00001.
microscope micrographs were acquired and depicted a
tightly formed HUVEC monolayer (Fig. 4(b)). Next, we
assessed changes to the membrane structure by monitor-
ing changes in transendothelial electrical resistance. Treat-
ment with TMZ@GSLN resulted in an 18% decrease in
TEER 15 min following treatment before trending towards
original values (Fig. 4(c)). TEER values of the vascular
model over time suggest that GSLN were able to reversibly
modulate the vascular barrier, supporting GSLN ability to
interact with endothelial cells for the purpose of delivering
TMZ across a vascular barrier in vitro.
Following a 1 h treatment with TMZ@GSLN, samples
from the lower chamber were collected and assessed using
spectrophotometry to determine the amount of drug that
diffused through the vascular barrier. Our results indicate
10% of the free TMZ solution was able to diffuse through
the monolayer while TMZ@GSLN was able to transport
approximately 30% of the loaded drug across the endothe-
lial monolayer, a 60% increase in drug diffusion compared
to free drug (Fig. 4(d)). This effect could be attributed
to the highly lipophilic nature of ghee and lipid-based
nanoparticles, facilitating the passage across the barrier.44
Additionally, ghee may enable dynamic changes to the
endothelial cells accelerating drug diffusion across the
monolayer.
4. CONCLUSION
The primary objective of this study was to investigate
the feasibility of using SLN synthesized from ghee as
a drug delivery vector. Our results illustrate that the
use of ghee for the manufacturing of SLN could pro-
vide high encapsulation efficiency with minimal cytotoxic
effects on healthy cells. Specifically, we demonstrate that
GSLN provide greater than 60% entrapment efficiency
of a chemotherapeutic payload. Additionally, our results
showcase GSLN alone provided a statistically significant
increase in viability for endothelial cells while substan-
tially minimizing proliferation of glioblastoma cancer cells
when loaded with TMZ. As a result of favorable biocom-
patibility properties and high entrapment efficiency, GSLN
can be exploited for other drug delivery applications as an
alternative to traditional methods.
Acknowledgments: This work was supported by the
Cullen Trust Foundation.
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Received: 11 November 2015. Accepted: 24 November 2015.
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