Biomedicine & Pharmacotherapy 93 (2017) 561–569

Original article

Endocytic pathways of optimized resveratrol cubosomes capturing intohuman hepatoma cells

Hend Mohamed Abdel-Bara,*, Rania Abd el Basset Sanadb

aDepartment of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, University of Sadat City, Menoufia, EgyptbDepartment of Pharmaceutics, National Organization of Drug Control and Research, Giza, Egypt

A R T I C L E I N F O

Article history:Received 16 April 2017Received in revised form 17 June 2017Accepted 23 June 2017

Keywords:ResveratrolCubosomesCytotoxicityEndocytic pathwaysCaveolae

A B S T R A C T

Resveratrol (RSV) is a natural polyphenolic compound with high affinity to hepatocytes. It has numerousbenefits as anticancer, antioxidant, immunomodulatory and cardioprotective actions. Nevertheless, RSVtherapeutic applications are hindered by its low solubility, light sensitivity and extensive first-passmetabolism. Cubosomes are colloidally stable dispersed liquid crystalline nanoparticles. The incorpo-ration of RSV into cubosomes could overcome some of its physicochemical limitations. A Design-Expert1

software was applied to optimize cubosomes in terms of particle size and encapsulation efficiency (EE%).The used model proved its suitability in predicting optimum cubosomal size. The prepared cubosomesshowed an enhanced HepG2 cytotoxicity except at particle size of �20 nm. Different endocytic pathwaysmechanisms as macropinocytosis, clathrin-mediated endocytosis and caveolae-mediated endocytosiswere identified in the cellular uptake of RSV cubosomes depending on particle size. Caveolae-mediatedtransport was shown to have a significant effect on RSV cubosomes internalization efficiency andcytotoxicity.

© 2017 Elsevier Masson SAS. All rights reserved.

Available online at

ScienceDirectwww.sciencedirect.com

1. Introduction

The successful ability of nanocarriers’ applications in cancertherapy necessitates efficient cellular internalization. Hence,understanding nanosystem-cell interactions and the mechanismof intracellular trafficking is crucial [1]. In general, endocyticcellular uptake is governed by two types of competitive energy thataffect the rate and amount of nanoparticles internalization. Thefirst one is the binding energy between ligands and receptors. Thisenergy denotes the interaction and the diffusion betweenreceptors and their ligands. The second force is the thermodynamicdriving force. This force represents the amount of free energyessential to internalize nanocarriers inside the cells [2]. Obviously,paracellular and transcellular transport are the two main pathwaysfor nanocarriers’ uptake [3]. Paracellular transport is a passivediffusion mechanism via the intercellular spaces and tightjunctions. Previous studies reported that negatively chargedparticles have no influence on disturbing the tight junctions [4].Consequently, negatively charged nanosystems could be

* Corresponding author at: Department of Pharmaceutics and IndustrialPharmacy, Faculty of Pharmacy, University of Sadat City, Abdel Moneim Riad St,Sadat City, Menoufia, Egypt.

E-mail address: hend.abdelbaar@pharma.asu.edu.eg (H.M. Abdel-Bar).

http://dx.doi.org/10.1016/j.biopha.2017.06.0930753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

implemented in transcellular endocytic pathway only. Trans-cellular endocytosis includes macropinocytosis, clathrin-mediatedendocytosis, caveolae-mediated endocytosis or caveolae- andclathrin-independent endocytosis [5]. These different endocyticpathways differ in the size it could internalize and the fate of thepenetrated nanocarrier [6]. Generally, the relevant applications ofnanocarriers are closely related to their size, shape, and surfaceproperties which with the membrane dynamics would affect thecellular uptake [7]. Particle size could be considered one of themajor factors that affect cellular uptake mechanism and biodis-tribution of nanocarriers [8]. Nanocarriers with size less than200 nm have superior extravasation and accumulation into tumorsby the enhanced permeability and retention effect [9]. Nano-systems rigidity seems also to influence the cell entry pathway;rigid nanoparticles are more susceptible to phagosomes formation,macropinocytosis is more involved in soft nanocarriers uptake,clathrin-mediated mechanism for stiff nanoparticles, and finallymultiples mechanisms are implicated for nanosystems withintermediate elasticity [10].

Among various nanocarrier systems, cubosomes have gained agreat interest either in drug and/or vaccine delivery [11].Cubosomes are colloidally dispersed nanostructured liquid crys-talline nanoparticles characterized by a three-dimensional cubicinner structure [12]. They are lipid bilayer-based particulates built

562 H.M. Abdel-Bar, R.A. el Basset Sanad / Biomedicine & Pharmacotherapy 93 (2017) 561–569

on self-assembled systems disseminated in an aqueous environ-ment. Cubosomes are composed of biodegradable and biocompat-ible lipids as glyceryl monooleate (monoolein), mixtures of soyphosphatidylcholine and glycerol dioleate which can be dispersedin water by the use of stabilizers and/or surfactants as TPGS (D-alpha-tocopheryl poly (ethylene glycol) 1000 succinate), Tween 80or Pluronics. By virtue of their structure, they offer more appealingcharacteristics than liposomes such high stability and mechanicalrigidity [13].

Resveratrol (RSV) is a polyphenolic compound naturally existsin grapes and peanuts which exhibits anticancer, antioxidant,immunomodulatory and cardioprotective actions. RSV is supposedto constrain different tumorigenesis stages; initiation, promotionand progression [14]. Previous reports had demonstrated the highaffinity of RSV for hepatocytes [15,16]. Unfortunately, RSV suffersfrom low aqueous solubility, extensive first pass metabolism, andisomerization to the inactive cis-isomer by light exposure [17].Drug encapsulation in nanocarriers could avert these challengeswhere the bioavailability will depend on the nanosystem ratherthan physicochemical properties of the drug [3]. Due to itsimmense pharmaceutical uses, RSV had been extensively studiedin various nanoformulations. Previous studies had demonstratedthe incorporation of RSV in different nanocarriers as polymericnanoparticles, micelles, liposomes and solid lipid nanoparticles.These nanocarriers afford numerous benefits as improved aqueoussolubility, controlled drug release, improved stability and bio-availability as well as enhanced cytotoxicity [18–20].

Hence, the aim of this study was the tuning of RSV cubosomescharacters to improve RSV cellular uptake and cytotoxicity againstHepG2 human hepatoma cells. This study also focused on trackingthe different endocytic pathways that RSV cubosomes could becaptured into cells.

2. Materials and methods

2.1. Materials

Resveratrol (RSV) was obtained from Xi'an Sonwu Biotech Co.,Ltd., China; glyceryl monooleate (GMO), poloxamer 407 (P407),RPMI-1640 medium, heat-inactivated fetal bovine serum (FBS),penicillin, streptomycin, L-glutamine, sulforhodamine B (SRB), Trisbuffer, trypsin-EDTA, amiloride, chlorpromazine, nystatin andglacial acetic acid from Sigma–Aldrich Company, St. Louis U.S.A;acetonitrile, ethanol and methanol (HPLC grade) from Riedel-deHaenGmbh, Germany.

2.2. Experimental design

A three-factor, each at three-level, Box-Behnken design wasexploited to statistically optimize the variables for the preparationof RSV nanocubic vesicles using Design Expert1 software (Version9.0.6.2, Stat-Ease Inc. Minneapolis, MN, USA). The influence ofthree independent variables namely: glyceryl monooleate/polox-amer 407 (GMO/P407) ratio (X1), homogenization speed (X2) andhomogenization time (X3) on the dependent variables, particlesize and entrapment efficiency (EE%) were assessed. The influence

Table 1Box-Behnken design for the optimization of resveratrol cubosomes.

Independent variables Levels

Low Medium

GMO/P407 ratio 2.5:1 5.75:1

Homogenization speed (rpm) 11000 15000

Homogenization Time (min) 1 5.5

of different factors on the characters of RSV cubosomes wasinvestigated to obtain RSV with particle size range of 20–100 nmand maximum EE%. All experiments were performed in triplicateto validate the final predicted results compared to the experiments[21]. The optimized formulations were selected depending on thecalculated desirability and were then prepared in triplicate tocheck the validity of the evolved statistical model before beingsubjected to further investigations. The different levels of theindependent factors used in the experiment design and thedependent responses were clarified in Table 1.

2.3. Preparation of RSV cubosomes

Briefly, GMO and P407 (5%w/w) were molten in a water bath at70 �C. RSV was accurately weighed and added to the moltenmixture and vortexed to ensure thorough mixing. The mixture wasadded gradually into deionized water (95%w/w) at 60 �C [22]. Theobtained dispersion was subjected to different homogenizationspeed and time using high-speed homogenizer (Unidrive X1000DHomogenizer, CAT Scientific, USA). The concentration of RSV in theprepared cubosomes was kept 1%w/w for all formulations. Theprepared nanocubic vesicles were allowed to cool gradually toroom temperature and kept in screw-capped amber glass vials forfurther investigations.

2.4. Characterization of the prepared RSV cubosomes

2.4.1. Particle size and zeta potential measurementsParticle size, polydispersity index (PDI) and zeta potential of the

prepared RSV cubosomes were measured by photon correlationspectroscopy using Zetasizer (Malvern Instruments Ltd., UK). Themeasurements were determined at 25 �C using an angle of 90�.

2.4.2. Determination of entrapment efficiencyThe entrapment efficiency percent (EE%) of RSV in the prepared

cubosomes was determined by centrifugation at 2400g for 3 h at4 �C using Ultra Centrifuge (Jouan, France). The pellets weredissolved with ethanol and RSV content was determinedspectrophotometrically at 306 nm (UV–vis double beam spectro-photometer Shimadzu 2450, Japan). An empty cubosomes wereused as a blank. EE% was calculated according to the followingequation [23]:

EE ¼ amount of entraped RSVTotal amount of RSV added

� 100 ð1Þ

2.4.3. Transmission electron microscopy (TEM)The morphology of the optimized RSV cubosome was examined

using TEM (JEOL, JEM-HR-2100, Japan). The cubosome was dried ona carbon coated copper 300-mesh grid and was stained with 1%phosphotungstic acid before the examination.

2.4.4. In vitro RSV release studyThe in vitro release of RSV from the optimized cubosomes was

assessed, in the dark, using dialysis method [24]. An accuratelyweighed RSV cubosomes (0.5 g) were placed into presoaked

Dependent variables

High Response Constraints

9:1 Particle size (nm) 20–1001900010 Entrapment efficiency (%) maximize

Table 2The observed responses in three-factor each at three-level Box-Behnken design for the formulation of RSV loaded cubosomes.

Run X1GMO: P407 ratio

X2Homogenization Speed (rpm)

X3Homogenization Time (min)

Y1Particle size (nm)

Y2Entrapment Efficiency (%)

1 5.75 19000 1 116.66 � 1.21 91.00 � 2.412 5.75 11000 1 190.00 � 2.55 92.66 � 1.653 2.5 11000 5.5 162.00 � 1.65 95.00 � 3.244 5.75 15000 5.5 98.00 � 2.34 89.66 � 1.245 5.75 15000 5.5 98.33 � 2.77 91.00 � 3.216 9 15000 10 45.00 � 1.55 96.66 � 1.217 5.75 11000 10 155.00 � 2.31 96.00 � 2.218 2.5 15000 1 106.66 � 1.39 93.66 � 3.219 5.75 19000 10 22.33 � 1.26 96.33 � 1.2110 5.75 15000 5.5 98.00 � 3.21 89.66 � 2.3111 5.75 15000 5.5 97.00 � 4.12 90.20 � 1.9412 2.5 15000 10 121.00 � 2.33 97.10 � 1.4113 5.75 15000 5.5 98.00 � 1.41 90.66 � 1.3214 9 11000 5.5 195.00 � 2.99 92.00 � 1.4115 9 15000 1 162.00 � 3.11 90.66 � 2.1116 9 19000 5.5 61.00 � 2.41 91.66 � 2.3417 2.5 19000 5.5 44.00 � 3.91 91.67 � 2.74

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dialysis bag (Spectra/Por1 CE cut off MWCO12–14 kD, flat width10 mm and diameter 6.5 mm, USA). The dialysis bag was attachedto the shaft of the USP dissolution testing apparatus type I (HansonResearch Corporation, USA) containing 150 mL PBS (pH 7.4). Theshaft was rotated at 50 rpm � 0.1 at 37 �C � 0.5 and the experimentwas conducted for 30 h. At predetermined time intervals, analiquot of 1 mL was withdrawn and replaced with the same volumeof fresh dissolution medium. Samples were diluted with ethanol ina ratio of 1:1 (v:v) and quantified for drug content byspectrophotometric analysis at 306 nm [24]. An empty cubosomeswere used as a blank.

2.4.5. Stability studies

2.4.5.1. Thermal stability. Thermal stability of the optimized RSVcubosomes was conducted in dark at 4 and 25 �C for 28 days. At day7, 14, 21 and 28 samples were visually inspected and entrapmentefficiency, particle size, zeta potential were determined [25].

2.4.5.2. Freeze-Thaw stability. The optimized RSV cubosomes weresubjected to three repeated cycles of freezing at �80 �C for twodays followed by storage at 25 �C for another two days. Samplesthen were analyzed in terms of entrapment efficiency, particle sizeand zeta potential [25].

2.4.6. In vitro cytotoxicity assessment of the optimized RSV cubosomesIn vitro antitumor activities of the both the empty and

optimized RSV nanocubic vesicles as well as RSV solution in PBS(pH 7.4) were assessed on human hepatoma HepG2 cell line. Thecells were grown as monolayers in RPMI-1640 medium containing100 units/mL of penicillin, 100 mg/mL of streptomycin and 1% L-glutamine. The medium was supplemented with 10% heat-inactivated FBS at 37 �C under a humidified atmosphere containing5% CO2 (Thermo Scientific Forma1, Germany). The grown cellswere seeded in 96-well microtitre plates at a concentration of 104/well and incubated for 24 h at 37 �C with 5% CO2. Afterward, cellswere incubated with serial concentrations of cubosomes in RSVrange of 0.78–25 mg/mL at 37 �C for 24 h. The cytotoxicity wasmeasured using sulforhodamine B (SRB) assay. Briefly, cells werefixed with 10% w/v trichloroacetic acid for 1 h at 4 �C then stainedwith 0.4% w/v SRB for 15 min. The formed dye complex wassolubilized with 10 mM unbuffered Tris-HCl. The optical density(OD) of each well was assessed spectrophotometrically at 540 nmusing a plate reader (ChroMate-4300, FL, USA). The absorbance of

control untreated cells was considered as 100% proliferation. The50% inhibitory concentration (IC50) was assessed graphically [26].

2.4.7. Evaluation of transport across HepG2The cellular uptake of nanoparticles is an energy-dependent

process highly affected by time and concentration. Therefore, theeffect of the optimized cubosomes in different RSV concentrationsand incubation time on the intracellular uptake efficiency wouldbe evaluated as follow:

2.4.7.1. Effect of time. The exponentially grown HepG2 cells wereincubated with 100 mL (containing 20 mg/mL RSV) of differentcubosomes for 0.5, 1, 2 or 4 h. Cells were washed three times withice-cold PBS to remove untaken cubosomes. Cells were trypsinizedand lysed using 0.1% sodium dodecyl sulfate, 0.1 M sodiumhydroxide and 2% sodium carbonate [27]. The cell homogenatewas centrifuged at 1530g for 10 min at 4 �C. The obtainedsuspension was mixed with an equal volume of acetonitrile andcentrifuged at 860g for 30 min. The clear supernatant was used fordrug quantification. RSV concentration was assayed using HPLC(Agilent 1100, Germany) equipped with G 1311 A quaternary pumpand UV detector (VWD-G1314 A). A reverse phase column (ACE 5-C18, 250 � 4.6 mm, 5 mm) was used at 25 �C. RSV was eluted with amobile phase consisted of methanol: water (containing 0.05%acetic acid) in ratio 52:48 (v:v) at a flow rate 1 mL/min. The UVdetector was set at 310 nm [24].

2.4.7.2. Effect of concentration. The seeded HepG2 cells wereincubated with 100 mL of different concentrations of the preparedcubosomes (1, 5, 10 and 20 mg/mL RSV) for 4 h [1]. Cells werewashed three times with ice-cold, trypsinized and lysed by thesame procedures as mentioned above. The RSV concentration wasassayed using HPLC as previously described.

2.4.7.3. Effect of energy variation. The grown HepG2 cells wereincubated at 4 �C for 2 h [3]. Subsequently, cells were incubatedwith 100 mL of the prepared RSV cubosomes (containing 20 mg/mLRSV) for 4 h. Cells were treated as previously mentioned todetermine RSV cellular concentration.

2.4.8. Tracking pathway of cellular uptakeThe influence of different endocytic inhibitors on the cellular

uptake was assessed. Briefly, the seeded HepG2 cells wereincubated separately with chlorpromazine hydrochloride (5 mg/mL) as an inhibitor of clathrin-mediated endocytosis, nystatin

564 H.M. Abdel-Bar, R.A. el Basset Sanad / Biomedicine & Pharmacotherapy 93 (2017) 561–569

(50 mg/mL) as caveolae-mediated endocytosis inhibitor andamiloride (13.3 mg/mL) as macropinocytosis inhibitor for 1 h at37 �C [1]. Afterward, cells were treated with 100 mL of different RSVcubosomes (containing 20 mg/mL RSV) for 4 h and RSV cellularconcentration was determined as previously described.

2.5. Statistical analysis

Results were expressed as the mean of three replicates �standard deviation (SD). For comparing different parametersbetween groups, one-way analysis of variance (ANOVA) followedby Tukey HSD test was conducted using SPSS 18 (Chicago, U.S.A.).The differences were statistically significant at a probability level(p) less than 0.05.

3. Results and discussion

For the response surface methodology using Box-Behnkendesign experiment, a total of 17 runs were implemented toinvestigate the influence of GMO: P407 ratio, homogenizationspeed and time on RSV cubosomes particle size and EE%. Suitablestatistical models for RSV cubosomes optimization were chosenbased on the adjusted R2, predicted R2 and the lowest predictedresidual sum of squares (PRESS) [28].

Table 2 shows the 17 experiment runs with three independentvariables each at three levels and the obtained particle size and EE%as responses. The 2FI and quadratic models were selected as thestatistical models describe the effect of different RSV cubosomesformulation on particle size and EE% respectively since they hadthe least PRESS values (Table 3).

3.1. Effect of formulation variables on the particle size (Y1) of theprepared RSV cubosomes

Nanocarrier size is one of the most factors affecting cellularuptake and biodistribution. The mean particle size of the preparedRSV cubosomes ranged from 22 � 1.26 to 195 � 2.99 nm (Table 2).The polydispersity index PDI of all formulae was <0.2 whichindicated the narrow homogenous particle size distribution of theprepared RSV cubosomes [29]. Fig. 1 illustrates the responsesurface for the influence of different formulation variables onparticle size. Equation (2) shows the effect of the different variableon RSV cubosomes particle size:

Particle size = +110.00 + 3.67 � X1-57.25 � X2-29.00 � X3-32.83*X1� X3 � 14.83 � X2 � X3 (2)

Where X1 is GMO: P407 ratio, X2 is homogenization speed and X3is homogenization time.

The ANOVA results revealed the insignificant effect of GMO:P407 ratio on particle size in the tested concentrations (p > 0.05).Furthermore, the negative sign of X2 and X3 coefficients indicateda significant inverse relation between homogenization speed andtime and particle size (p < 0.05). High homogenization speed andtime could amplify mechanical and hydraulic shear that breakcubic gel structure into nanocubic vesicles with smaller particlesize [30,31].

Table 3Results of regression analysis for responses Y1 (particle size) and Y2 (EE%).

Response Model Adequate precision R2 A

Particle size 2FI 19.695 0.9412 0EE% quadratic 14.590 0.9701 0

3.2. Effect of formulation variables on the EE% (Y2) of the prepared RSVcubosomes

The influence of different formulation variables on EE% could bedepicted from Table 2 and Fig. 2 according to the followingequation:

Entrapment efficiency = + 90.24 � 0.80 � X1 �0.62 � X2 + 2.26 �X3 + 0.75 � X1 � X2 + 0.64 � X1 � X3 + 1.43 � X12 + 0.91 � X22 +2.85 � X32 (3)

Where X1 is GMO: P407 ratio, X2 is homogenization speed and X3is homogenization time.

Negative signs of both GMO: P407 and homogenization speedindicate an antagonistic effect on EE%. At high GMO: P407 ratio, thesurfactant amount is not sufficient to solubilize high amount ofRSV into the lipid vesicles [32]. Moreover, increasing homogeniza-tion speed decreases the contact between RSV and GMO. On thecontrary, a direct relation between homogenization time and EE%could be observed. As by increasing contact time between drug andcubosome components, EE% could be improved.

3.3. Optimization of RSV cubosomes

To verify the developed models, five formulations, selectedaccording to high desirability, were prepared as a checkpoint.Table 4 shows the composition, predicted and experimentalresponses. The linear correlation plots between experimental andpredicted values for both responses showed high R2 (0.998 and0.979 for particle size and EE% respectively). Hence, the model wassuitable for studying and predicting the most suitable conditionsfor the fabrication of RSV negatively charged cubosomes of sizerange 20–100 nm. Thus 5 formulations were subjected for furthercharacterization.

3.4. Zeta potential of the optimized RSV cubosomes

The selected RSV cubosomes had zeta potential values rangedfrom �17.32 � 2.71 to �32.12 � 1.41 mV (Table 4). Where thecubosomes with the highest GMO ratio (highest fatty acid content)having the highest charge. The negative charge was possiblyimparted by the free fatty acids present in the lipid [33].

3.5. Transmission electron microscope

The TEM image of F2 is depicted in Fig. 3A. Scattered nanocubicvesicles with a diameter range of 40–50 nm in agreement with thedroplets size results determined by DLS. Spherical globulesappearing in the field might be molten cubosomes under thehigh-energy beam of the TEM [30]. Moreover, P407 that did notincorporate into the cubosomal matrix could participate in theformation of mixed micelles [23].

3.6. In vitro RSV release

Fig. 3B illustrates release profile of RSV from differentcubosomes over 30 h. By inspecting release profiles, an obviouseffect of GMO: P407 ratio could be discernible. Cubosomes areconsidered as a biphasic burst release delivery system with

djusted R2 Predicted R2 SD %CV p-value

.9144 0.8176 14.74 13.40 <0.0001

.9401 0.8676 0.63 0.68 <0.0001

Fig. 1. Response 3D plots for the effect of homogenization speed (X2) and time (X3) (A), effect of GMO: P407 (X1) and homogenization time (X3) (B) on RSV cubosomesparticle size.

Fig. 2. Response 3D plots for the effect of GMO: P407 (X1), homogenization speed (X2) and time (X3) on RSV cubosomes entrapment efficiency.

H.M. Abdel-Bar, R.A. el Basset Sanad / Biomedicine & Pharmacotherapy 93 (2017) 561–569 565

diffusion release mechanism from the cubic-phase matrix [34].The prepared formulae showed a biphasic release pattern with aninitial burst release for 6 h. This might be attributed to theinadequately adsorbed RSV on the cubosome surface. The secondsustained phase which prolonged for 30 h could be due to thearchitecture of cubosome matrix that assists as a rate controllingmembrane, hampered RSV release [34]. Furthermore, Nanocubicvesicles may exist in different types according to P407 concentra-tion. At low P407 concentration, cubic particles have Pn3 m (cD)forms where most of the P407 stick to the cubosomes surface. Onthe contrary, P407 incorporated in the cubic bulk matrix byincreasing its concentration and cubosomes exists in Im3m (cP)patterns. It is to be noticed that cubosomes with cD structure havesuperior drug release efficiency than cP cubosomes [35]. Moreover,

Table 4The experimental and predicted characters of the optimized RSV cubosome formulatio

Formula code GMO:P407 ratio

Homogenization Particle size (nm)

speed (rpm) time (min) Exp. Pred. % pred. er

F1 5.75 19000 1.00 97.65 96.58 1.09

F2 8.00 16000 9.83 45.63 44.47 2.53

F3 5.75 19000 8.17 28.61 27.95 2.28

F4 8.60 16000 7.33 76.57 75.35 1.59

F5 2.50 19000 5.50 50.14 49.08 2.11

at the same GMO: P407 ratio, the increase in particle size decreaseparticles surface area in contact with dissolution medium andtherefore caused a restriction in dissolution efficiency. Concerningthe kinetics of RSV release from the optimized cubosomes, thehighest R2 was >0.99 obtained from diffusion model indicating acontrolled diffusion release mechanism. The release of RSVoccurred via multiple consecutive steps where the drug isdissolved within the matrix of the cubosomes then RSV diffusesthe system via lipid/water interface [36].

3.7. Stability studies of RSV cubosomes

All the prepared RSV cubosomes reserved its original appear-ance with no signs of separation or precipitation. No significant

ns.

EE% Zeta potential (mV) �SD IC50 (mg/mL)

ror Exp. Pred. % pred. error

92.35 91.11 1.34 �22.64 � 1.51 12.3596.47 95.64 0.85 �32.12 � 1.41 5.193.89 92.85 1.10 �25.47 � 2.12 16.893.1 92.32 0.83 �30.41 � 2.54 9.293.45 92.01 1.53 �17.32 � 2.71 6.3

Fig. 3. TEM micrograph of RSV cubosome (A), in vitro resveratrol release from different cubosomes (B). Data are the mean of three determinations � SD.

Fig. 4. The effect of freeze-thaw cycles and storage at; 4 �C (A) and 25 �C (B) on entrapment efficiency, 4 �C (C) and 25 �C (D) on particle size, 4 �C (E) and 25 �C (F) on zetapotential. Results are the mean of three � SD.

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difference in entrapment efficiency, particle size or zeta potentialcould be observed after storage at either 4 or 25 �C up to 28 days(Fig. 4). Moreover, RSV cubosomes exhibited no significant changein entrapment efficiency, particle size or zeta potential afterfreeze-thaw stability study as evidence by Fig. 4.

3.8. In vitro cytotoxicity of the optimized RSV cubosomes

Fig. 5A illustrates high cell viabilities more than 80% with all thetested empty cubosomes indicating that plain formulations had nocytotoxicity [37]. The deterioration in HepG2 human hepatomacells viability incubated with both RSV cubosomes and solutiondemonstrated a rapid decline in viability of cells in a concentra-tion-dependent manner (Fig. 5B) [38]. It was obvious that thecalculated IC50were also particle size dependent, with the superiorantitumor activity of all RSV cubosomes over solution (IC50

14.5 mg/mL) except in formulation F3 with the size � 28.61 �1.51nm Table 4. Moreover, cubosomes with a particle size of 45–50 nmshowed superior cytotoxic activity over other formulations.Therefore, the particle size could affect the in vitro cytotoxicityof RSV cubosomes.

3.9. Factors affecting RSV cubosomes internalization efficiency

Cellular recognition of the fabricated nanocarriers has beenreported to be mainly size and surface chemistry dependent. It waspreviously mentioned that the cellular uptake of negative nano-carriers occurs mainly via endocytosis [4]. In addition, the rate ofendocytic uptake of nanoparticles with size less than 200 nm isstrongly size dependent. Accordingly, the prepared RSV cubosomesinternalization will be evaluated in terms of time, concentrationand energy.

In the present study, the cellular uptake of RSV cubosomes byHepG2 was found to be increased after 4 h in a size dependentmanner. Longer incubation time was accompanied by betternanocarriers’ uptake (Fig. 6A) [39,40]. Similarly, the increase inconcentration from 1 to 20 mg/mL also went along with theincrease in cellular uptake by 12–17 fold especially in the case of F2and F5 (Fig. 6B). Finally, incubation of HepG2 cells at 4 �Csignificantly (p < 0.05) decreased the cellular concentration ofRSV (Fig. 6C). At low temperature, enzymes activity could bedeteriorated and the efficiency of energy production was alteredhence inadequacy of cellular energy for uptake. Accordingly, thenanocarriers’ cellular uptake was an active energy-dependentprocess. The energy was produced by the mitochondrial aerobicoxidation and glycolysis in the cytoplasm [41].

Fig. 5. Cell viability after exposure to different empty cubosomes (A) and differendeterminations � SD.

3.10. Tracking pathway of cellular uptake

Cellular internalization is assumed to occur by the formation ofa protein coat vesicle on the cytoplasmic outer side of the cellmembrane [42]. Amiloride is a macropinocytosis inhibitor as itinhibits Na+/H+ exchange in the plasmatic membrane essential formacropinocytosis [43]. When added to the cells it inhibited theuptake of F1 (97.65 nm) significantly by 27% (p < 0.05). Chlor-promazine hydrochloride hinders of clathrin-mediated endocyto-sis by promoting clathrin agglutination in late endosomesobstructs the endocytosis sag [44]. Uptake of the formulationsF2, F3, F4 and F5 (28–76 nm) had significantly declined by 20–30%in the presence of chlorpromazine hydrochloride (p < 0.05)(Fig. 6D). Finally, nystatin constrains caveolae-mediated endocy-tosis by inhibiting caveolae-mediated endocytosis due to choles-terol segregation in the plasma membrane hinders sag vesiclesformation [43]. Accordingly, the treatment of cells with nystatinprior to incubation with different RSV cubosomes resulted in asignificant diminish in RSV uptake in cubosomes F2, F4 and F5(p < 0.05) (Fig. 6D). It is to notice that these three cubosomes had aparticle size in the range 45–76 nm.

Cellular uptake mechanism was clear to be a particle sizedependent process. Cellular penetration of F1 with a high particlesize (97.65 � 4.50 nm) was mainly followed micropinocytosis only,endocytosis was abolished for particles with size more than 80 nm[2]. F3 with the least evaluated size (�28 nm), was infiltrated intocells by clathrin-mediated endocytosis mechanism only and hadthe least efficacy. Particles with size less than 40 nm are unable toproduce sufficient free energy to enwrap to the cellular membranesurface which may avert endocytosis [2]. Finally, formulations F2,F4 and F5 with size range 45–76 nm were internalized by bothclathrin- and caveolae-mediated endocytosis. The dual uptakepathways contributed with cubosomes in the size range 45–76 nmcould explain their superior cytotoxicity. In addition, formulationsF2 and F5 with 45–50 nm in diameter showed lower IC50 than F4(76 nm). These results are in good agreement with previous resultswhich showed that nanocarriers with size �45 nm had the highestcellular internalization rate [9].

Correlating the cellular uptake with the trafficking resultsrevealed a poor cell penetration in case of F1 and F3 cubosomeswhere only one cellular uptake mechanism, either macropinocy-tosis or clathrin-mediated endocytosis, was involved in theinternalization process. In macropinocytosis process, actin whichis the most abundant cellular protein initiates the folding ofmembrane projections with the plasma membrane and construct-ing macropinosomes. Unfortunately, macropinosomes vesiclescould route the captured nanocarriers either to lysosomes or

t resveratrol cubosomes (B) on HepG2 hepatoma. Data are the mean of three

Fig. 6. Cellular uptake of different RSV cubosomes (A) at different time intervals, (B) at different concentrations, (C) after incubation at 4 �C and (D) after the influence ofdifferent endocytosis inhibitors. Data are the mean of three determinations � SD.

568 H.M. Abdel-Bar, R.A. el Basset Sanad / Biomedicine & Pharmacotherapy 93 (2017) 561–569

efflux its payload to surface again [45]. Moreover, clathrin-mediated endocytosis exists in the presence of clathrin proteinsthat occupy only 2% of the plasma membrane. Clathrin proteinsassociate to form basket shaped vesicles. These vesicles carry theinternalized nanocarriers to early endosomes and associated withprelysosomal vesicles to reach late endosomes and degradationdue to the fusion of endosomes with the acidic lysosomes [10].Therefore, nanocarriers are susceptible to lysosomal degradationafter its internalization by the aforementioned mechanisms.

However, F2, F4 and F5 cubosomes, which were internalized byboth clathrin- and caveolae-mediated endocytosis, have gotimproved cellular uptake results. It was previously reported thatcaveolae are membrane curvature with diameter range from 60 to80 nm with 10–50 nm neck protrusion structure [46]. Therefore,the efficiency of nanocarriers’ uptake with a particle size smallerthan caveolae diameter is superior over larger particles [47]. Albeitcaveolae have the capability to expand their neck to uptake largernanocarriers, the internalization efficiency is diminutive [47].Moreover, caveolae dissociate from the plasma membrane andallocate their content to caveosomes during the caveolae-mediatedendocytosis. Caveosomes circumvents lysosomes and consequent-ly protect its nanocargo from lysosomal degradation [48,49].Consequently, the fabrication of nanosystems that can deliverdrugs into the cells by exploiting caveolae-mediated endocytosiscould have tremendous potential in drug delivery.

4. Conclusion

Concisely, cubosomes provide numerous benefits as ease ofpreparation, low cost, no need for organic solvent and improve RSVsolubility. It could be inferred that tailoring the different processparameters could optimize RSV cubosomes to improve its efficacy.The proposed cubosomes was able to control in vitro RSV releasefor 30 h. Efficient cytotoxicity and cellular uptake of RSV

cubosomes were attained with a particle size range 45–76 nmwhich was additionally internalized by caveolae-mediated endo-cytosis. Caveolae were found to improve RSV cubosomesinternalization efficiency and cytotoxicity as it could avertlysosomal attack. The internalization of RSV with caveolae-mediated endocytosis could help in achieving higher cellularconcentration and achieve better chemotherapeutic outcomes.Understanding the cellular internalization mechanism is a crucialphase in the optimization of cellular drug targeting strategies. Afinding which should be dealt with in depth.

Funding

This research did not receive any specific grant from fundingagencies in the public, commercial, or not-for-profit sectors.

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