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Colloids andSurfaces B: Biointerfaces 126 (2015) 426436
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier .com/ locate /colsur fb
Comparative real-time study ofcellular uptake ofa formulatedconjugated linolenic acid rich nano and conventional macro emulsionsand their bioactivity in ex vivomodels for parenteral applications
Debjyoti Paul a,b, Sayani Mukherjeea,b, Rajarshi Chakrabortyc, Sanjaya K. Mallickb,Pubali Dhara,b,
a Laboratory of Food Science & Technology, Food & Nutrition Division, University of Calcutta, 20 B Judges Court Road, Kolkata, West Bengal 700027,Indiab Centre for Research in Nanoscience & Nanotechnology,University of Calcutta, JD 2, Sector III,Salt Lake City, Kolkata, West Bengal 700098, Indiac Department of Biochemistry, University of Calcutta, 35,Ballygunge Circular Road, Kolkata, West Bengal 700019, India
a r t i c l e i n f o
Article history:
Received 10 September 2014
Received in revised form 5 December 2014
Accepted 26 December 2014
Available online 4 January 2015
Keywords:
-Eleostearic acidFlowcytometry
Nanoemulsion
Reactive oxygen species
Transmission electronmicroscopy
a b s t r a c t
The objective ofthe present study was to fabricate and monitor real-time, impact ofa stable conjugated
linolenic acid, -eleostearic acid (ESA) rich nanoemulsion (NE) formulation (d
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Table 1
Fatty acid composition of bittermelonseed oil as obtained by gas-liquid chromatography.
Test fat Fatty acid composition (w/w %)
C14:0 C16:0 C18:0 C18:1 C18:2 C18:3
Bitter melon seed oil 2.40.001 31.30.002 8.00.002 7.90.001 50.40.001
Results are expressedas meanSD for n=3 samples ofmethyl esters of fatty acids from theextracted andpurified bittermelonoil seeds.
balance (HLB) of 8-18 is required, while for aW/O formulation HLB
of 36 is required. However, in both instances of O/W and W/O
emulsion system, the ideal HLB value varies according to the oil
involved in the emulsification process [3]. Often, a single surfac-
tant is not sufficient to provide for the optimum HLB to stabilize
an emulsion formulation when the oil-excipient involved is con-
siderably non-polar. For this reason, co-surfactants are involved
withboth low and highHLB value. The optimumblend of suchsur-
factants to balance hydrophilic and lipophilic tendency results in
the formationof a stable emulsion formulation upon dilutionwith
water. On the basis of particle size distribution, long-termstorage-
related changes, thermodynamic stability andparticle structure of
dispersed oil and surfactant within an aqueous continuous phase,
O/W emulsion system can further be classified as conventional
emulsion (random non-homogenous particle size distribution),
nanoemulsion and microemulsions (homogenous nano-sized col-loidal dispersion systemswithd
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Fig. 1. Transmission ElectronMicroscopy (TEM) of theformulated ESANE:Particle size andappearance at differentmagnifications. (A)Mag= 32,000; (C) Mag = 15,600;
(B) Mag = 33,000; (D) Mag = 20,000.
the statistical analysis were carried outwith one-way ANOVA fol-
lowed by TukeyKramerpost hocanalysis (IBM SPSS Statistics 20).
Difference in results with P
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Fig. 2. (A)Time dependentuptake andmetabolism results of ESA NE & ESACE forlymphocytes(5105 cells perwell). Results areexpressed inmean fluorescence intensity
(MFI) SD (n=3). The results indicate theMFI emitted by NileRed conjugatedwith intact ESA rich triglyceride (TG). Metabolized TGsdue to cytoplasmic esterases activity
(inside lymphocytes) leading to release of free fatty acids show quenched MFI due to the extremely sensitive fluorescent expression of Nile Red under core hydrophobic
conditions. (B) Flow cytometric representational analysis of Nile red conjugated ESA-NE and ESA-CE uptake andmetabolism. Lymphocyte population were electronically
gated to study and compare the uptake and metabolism of ESA rich colloidal systems (NE and CE) via transcellular pathway (lymphocytes being non-adherent cells) on a
time dependentmanner. Results are indicatedas MFI andwere carried out in triplicates for 5105 PBMCs perwell.
conditions of 60%5% relative humidity without shaking. The
particle size, zeta potential data of the system for 12 weeks
has been recorded as Table 2. However, due to physicochem-
ical conditions such as Oswald ripening, gravitational pull and
Laplaces pressure adversely affecting the stability of the CE
system had showed signs of cracking in 1 week of storage
(Table 2).
The zeta potential was found to be >25mV as per the electro-
acoustic measurement for the ESA NE system during the entire
storage period. The experimental ESA NE had a zeta potential of
+33.72mV indicating good stability [14]. While the zeta potential
dropped to +17.01mV indicating mild stability after 12 weeks of
the storage period (Table 2).
3.3. Absorption of formulated ESA NE and CE systems
Time-dependentflowcytometric analysis of the PBMCs stained
with Nile Red for intracellular lipid content estimation and
grouped as per additional sample treatments of the formulated
stocksamples comprising 700MESANEand CEsystems upto4 hwas carried out and recorded (Fig. 2A and B). From the meanfluo-
rescence intensity paradigm corresponding to individual systems
of the freshly formulatedNEand CEof ESA, itwas revealed that the
absorptionofESANEwashigher thanESACE.Previousstudies have
established Nile red as a very specific intracellular triglyceride
fluorescent probe that shows no fluorescence inside the cyto-
sol or in the nuclear compartment. Thus, lipase metabolism of
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triacylglycerol (TAG) molecules conjugated with Nile red fluores-
cent dye will be quenched as compared to intact TAG molecules.
The meanfluorescence intensity data for 1-h, 2-h and 4-h for both
the ESA rich NE and CE systems indicated that the Nile red mean
fluorescence intensitywas significantlyhigher inside lymphocytes
for CE as compared to NE systems. The logical explanation of
this time-dependent paradigm observation could be attributed
to the fact that TAG metabolism of nano-sized oil droplets inside
lymphocytes is higher than micron-sized oil molecules inside
conventional emulsion systems. The results of the study also
provide empirical evidence that the absorption rate of ESA NE is
significantly higher thanESA-CE.
3.4. Reactive oxygen species measurement
Mean DCF fluorescence was proportionate to the increase in
intracellular (cellpopulationgated: lymphocytes) ROS.ROSgener-
ation for all the pre-treated test samples were comparedwith LPS
(pathogenic mitogen) induced control group and untreated sam-
ple/normal cells control grouptodeterminethe impact of different
doses of ESA-CE and ESA-NE on altering the excess ROS generated
due to activation of Toll-family of receptors [15] in LPS-treated
groups. Theintracellular ROSMFI(Mean Fluorescence Intensity) in
LPS-untreated group, served as the basal reference of ROS presentunder un-stimulated conditions for quantitative analysis.Here,we
found that 70M ESA NE reduced ROS level (proportionated withMFI) by 41% as compared to the positive control group (P
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Fig. 3. (A) Reactive Oxygen Species analysis (ROS) analysis of electronically gated lymphocyte population (5105 PBMCs per well) following pre-incubation with
variously dosed ESA rich colloidal systems and 1 (g/ml) LPS (pathogenic mitogen) challenge: A. NEGATIVE CONTROL: ROS analysis of the population of PBMC(thus, lymphocytes) unexposed to LPS challenge; B. POSITIVE CONTROL: ROS analysis of the population of untreated lymphocytes exposed to LPS induced chal-
lenge; C. 700M ESA CE: ROS analysis of the population of lymphocytes pre-incubated (1h) with 700M ESA CE followed by exposure to LPS challenge; D.700M ESA NE: ROS analysis of the population of lymphocytes pre-incubated (1h) with 700M ESA NE followed by exposure to LPS challenge; E. 70M ESACE: ROS analysis of the population of lymphocytes pre-incubated (1h) with 70M ESA CE followed by exposure to LPS challenge; F. 70M ESA NE: ROS analysis
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viscosity and interfacial tension as compared to short chain (SCT)
ormedium chain triacylglycerols (MCT). Yet onceformulated, LCTs
have higherphysical stability than the rest. Detailed perspicacious
modifications are thus required in conventional homogenization
techniques to formulate o/wnanoemulsions of LCTs [16]. We suc-
cessfullydevisedand incorporatedoptimization in formulating the
ESA NE in question to improve its stability and distribution. The
fabricated food-grade nanoemulsion system comprising non-toxic
excipients following high pressure homogenization was found to
be reflectingmonomodal distribution ranging frompolydispersity
(PdI)of 0.161 to0.247duringthe entirestorageperiodof 12weeks.
Although the systemwas stabilized withnon-ionic co-surfactants,
i.e. Tween 20 and Span 80, it was found to possess a high positive
charge of +33.72mV during the first week and the period dur-
ingbiological experimentationwith it as evinced by zeta potential
analysis. This aspect of the results is in agreement with the results
obtained by Li et al. [17] during their respective analysis of a PMF-
nanoemulsionsystemcomprisingnon-ionic surfactant (Tween20).
The zeta potential dropped to +17.2 only after 12 weeks of the
storage period and throughout the period indicated a stable for-
mulationdevoidof droplet creaming(i.e. throughout their volume,
uniformopaque appearancewas observed). The initial zeta poten-
tial analysis of the formulated ESA-NE up to 4 weeks indicated
results of good stability parameters to resist droplet aggregation[14]. On the contrary, the freshly prepared conventional emulsion
system of ESA with PdI of +12.4 indicated lower colloidal stability
prone to particle sedimentation. This parameter could well be the
genesis of the consequent fate of ESA-CE which showed creaming
at the end of 1 week with the oil component sedimenting by the
walls of the borosilicate container.
Initialstudiestoanalyzethe fateof triacylglycerol-emulsionsys-
tem in lymphocytes after 48h culture has shown that the activity
of lipoprotein lipase in hydrolysing oleic acid rich triacylglyce-
rols into free fatty acids is 10 units/mg protein [18]. Moreover,
recent studies have also shed some light on the mechanistic
pathway in the uptake of nanoparticles. Comparative studies in
non-phagocyticcellsbetweenuptakeofnanoparticles(d200nm, have revealed that internaliza-tion of the nanoparticles (d200nm, enter cells by caveolae dependent endocy-
tosis. It has also been established that processing of particles is
extremely rapid in clathrin-mediated endocytosis (although even
inclathrin-mediatedpathway, particlesizeswitha diameterbelow
100nm are even rapider than particles with diameter between
100 and 200nm) as such particles reach lysosomal compartments
within 30min of internalization by cells. On the contrary, caveo-
lae dependent endocytosis rarely reach lysosomal compartment
even after 4h of incubation. Now, since lipoprotein lipases are
present inside these lysosomal chambers, thedegree of hydrolysis
of triacylglycerols should logically be higher for clathrin-mediated
endocytosis for nanoparticles than caveolae dependent endocyto-sis forparticleswith a diameter greater than200nm [19]. Ourflow
cytometric results of non-phagocytic, non-adherent lymphocytes
thus are in excellent agreement with this experimentally estab-
lished theory.Nileredbeing anextremelyhydrophobicfluorescent
probe fluoresced till the TAG molecules accumulated inside the
lymphocytes would remain intact and will show a proportionate
increase in MFI. The flow cytometer acquired results thus cor-
responded to this un-metabolized, accumulated internalized TAG
fraction.
The MFI of Nile Red conjugated with the ESA-NE system dur-
ing the time-dependent analysis showed that both internalization
and metabolism were high during the first 30min of incubation
as compared to the ESA-CE system conjugated with Nile red. The
quenchedMFI of nile red associated with ESA-NE during the later
course of the incubation period could be attributed to the fact that
theclathrin-mediatedendocytosisof ESA-NE(particlesize lessthan
200nm)wasgettingrapidlymetabolizedandin theincreasedpolar
environment of free fatty acids inside the lysosomal chamber (site
of TAG metabolism). This paradigm of ESA-CE linked Nile red MFI
got conspicuously increased during the course of the incubation
period, aptly implying that thesystemgot internalizedvide caveo-
laemediated pathway, thereby getting accumulated at the cellular
periphery as they did not reach the lysosomal chambers before
4h. Thus, it can be logically asserted that the release and accumu-
lation of ESA being internalized vide clathrin-mediated pathway
were much higher in nanoemulsion systemthan internalizationof
thecaveolaemediated conventional emulsion system. It shouldbe
pointed out that non-specific macropinocytosis internalization of
theESA rich colloidalsystemscouldnot have been significant since
lymphocytes are non-phagocytic cells.Reactive oxygen species (ROS) have been identified as major
contributors towards the regulation and modulation of various
cellular processes including cellular survival, autophagy and cell
death.ROSreferstothefamilyof reactivemoleculesproducedin the
cells by the metabolism of oxygen capable of causing destruction
at higher concentrations. In addition to other non-radical species,
ROS are derived from oxygen metabolism and are present in cells
and tissues at low but measurable concentrations. This concen-
tration is dependent on the rate of production and its clearance
by inter-play of a multitude of antioxidative-enzyme systems and
non-enzymatic antioxidants. Moreover, the maintenance of this
redox homeostasis is crucial for the normal physiological func-
tioning of livingcells and tissues [20].
Investigativestudiesonidentifying thenodesof redox signallinghaveidentifiedmitochondria,endoplasmicreticulum,peroxisomes
and NADPH oxidase (NOX) as the major intracellular ROS gen-
erating sources, which includes singlet oxygen (1O2), superoxide
(O2), hydroxyl radical (OH), nitric oxide (NO) and hydrogen
peroxide (H2O2). In this context, the role of ROS in inducing
autophagy through a signalling cascade has also been established,
although precise mechanisms and effective therapeutic strategies
ofROS-regulatedautophagy remain stochastic [21]. Bacterialendo-
toxins such as lipopolysaccharide have been reported to induce
ROS generation in excess amounts and disrupting the redox-
homeostasis by activating Toll-like receptor 4 (TLR4)pathway and
simultaneouslyreducingperoxisome-activated receptor (PPAR)invitro [11,15]. This leadstoexcessnecrosisandatrophy of infected
cells, and this further accentuates intracellular pro-inflammatoryresponses overriding the anti-inflammatory counter balance.
Conjugated fatty acids such as ESA have been reported to
contributesignificantly incounter-balancingtheetiologicalparam-
eters causing oxidative stress in biological systems through a
multi-prong mechanism. ESA up-regulates intracellular PPARexpression [22] which in turn modulates the LPS mediated
of the population of lymphocytes pre-incubated (1h) with 70M ESA NE followed by exposure to LPS challenge. (B) Reactive Oxygen Species analysis (ROS) analysis ofPeripheral Blood Mononuclear cells (PBMC) population (5105 PBMCs per well) comprising lymphocytes, monocytes and granulocytes following pre-incubation with
variouslydosedESA rich colloidal systems and1 (g/ml) LPS(pathogenicmitogen)challenge:A. NEGATIVECONTROL: ROSanalysisof thepopulation of PBMCunexposedtoLPS challenge; B.POSITIVECONTROL:ROS analysis ofthe population of untreatedPBMCsexposedto LPSinducedchallenge; C. 700MESACE: ROSanalysisof thepopulationofPBMCs pre-incubatedfor 1h with 700M ESA CEfollowedby exposureto LPS challenge;D. 700M ESA NE:ROS analysis of thepopulation of PBMCs pre-incubated (1h)with 700M ESA NE followed by exposure to LPS challenge; E. 70M ESA CE: ROSanalysis of the populationof PBMCs pre-incubated (1h) with 70M ESA CE followed by
exposure to LPS challenge; F. 70M ESA NE: ROS analysis of the population of PBMCspre-incubated (1h) with 70M ESANE followed by exposure to LPS challenge.
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Fig. 4. Analysis of compromised (necrotized) electronically gated lymphocytes from a 100% population of 5105 PBMCs(per well) by PI staining due to 120min exposure
to 5g/ml LPS exposure in variously treated marked groups (analysis for each group were carried out in triplicates) with reflected results indicating statistical significanceofP
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formulations. In our previous studies also, on an in vivomodel we
had reported and explained the role of ESA NE in a reduced dose
to counter alloxan induced diabetes by providing for an additional
sourceof antioxidants to counter theabnormal ROSgenerateddue
to toxic accumulation of GSH-alloxan adducts [6]. This study thus
paves a way for further investigations on exploring the molecu-
lar mechanisms of the cellular mediators stimulated by the ESA
involved in mitigating parameters affected by excess intracellular
and exogenousROS.
In summary, a comparative analysis of a stable formulation
of ESA NE with an ESA CE formulation to enhance prophylaxis
against both endogenous and exogenous ROS in live cells is being
reported in this study. The findings of the present study havebeen
derived by tracking, interpreting and correlating the metabolic
fate of the ESA rich subject formulations in modulating molec-
ular parameters aggravated by a disturbed redox equilibrium in
non-phagocyticcells. Previousstudieshave alreadyhighlightedthe
therapeutic/prophylactic benefits of ESA as free fatty acids in vitro
and in silicomodels [25,26]. However,owing to thetransientnature
of free PUFAs such as ESA due to their proneness to oxidation,
it becomes a mounting challenge to deliver them in a stable un-
oxidized state through a stable formulation in vivo. In our reported
formulationusingcolloidalnanotechnologyhowever,we havesuc-
cessfully addressed this issue to fabricate a stable system of ESANE.A holistic picture of bioavailability of ESA and the implications
of its various doses to counter endogenous and exogenous ROS
in primary cells has been projected in this study. Moreover, since
the studies have been carried out only in human and human-like
(like that of rats) ex vivomodels, the clinical aspects of the ESA NE
& CE formulations too, if delivered parenterally at the optimized
dose becomes evident whichis partially in similar lines of previous
reports of other TAG rich parenteral emulsion systems [27].
Conflict of interest
The authors have no conflict of interest to report.
Acknowledgement
This work was supported by the Indian Council of Medi-
cal Research grant and Centre for Research in Nanoscience and
Nanotechnology,University of Calcutta infrastructure. Theauthors
would also like to acknowledge the immense technical help ren-
dered byMiss Urmila Goswami of JEOL India.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in theonlineversion,athttp://dx.doi.org/10.1016/j.colsurfb.
2014.12.046.
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