Comparative Real-time Study of Cellular Uptake of a Formulated Conjugated Linolenic Acid

7/25/2019 Comparative Real-time Study of Cellular Uptake of a Formulated Conjugated Linolenic Acid

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Colloids andSurfaces B: Biointerfaces 126 (2015) 426436

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Colloids and Surfaces B: Biointerfaces

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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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D. Paul et al. / Colloids and Surfaces B: Biointerfaces 126 (2015) 426436 427

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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430 D. Paul et al. / Colloids andSurfaces B: Biointerfaces 126 (2015) 426436

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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Comparative Real-time Study of Cellular Uptake of a Formulated Conjugated Linolenic Acid

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