Research Article In Vitro and In Vivo Enhancement of ...downloads.hindawi.com/journals/jnm/2015/967473.pdf · Islamic Azad University (IAUPS), Tehran, Iran Pharma Chemie Company,

Research ArticleIn Vitro and In Vivo Enhancement of AntitumoralActivity of Liposomal Antisense Oligonucleotides byCineole as a Chemical Penetration Enhancer

Hamid Reza Moghimi1 Farshad H Shirazi1 Mehdi Shafiee Ardestani2

Mohammad Ali Oghabian3 Mostafa Saffari45 and Jafar Sojoudi6

1School of Pharmacy Shahid Beheshti University of Medical Sciences Tehran Iran2Radiopharmacy Department Tehran University of Medical Sciences Tehran Iran3Research Center for Science and Technology in Medicine Tehran University of Medical Sciences Tehran Iran4School of Pharmacy Iran University of Medical Sciences Tehran Iran5Department of Medical Nanotechnology Faculty of Advanced Sciences amp Technology Pharmaceutical Sciences BranchIslamic Azad University (IAUPS) Tehran Iran6Pharma Chemie Company Tehran Iran

Correspondence should be addressed to Mostafa Saffari mostafasaffarygmailcom

Received 6 July 2015 Revised 18 August 2015 Accepted 19 August 2015

Academic Editor Abdelwahab Omri

Copyright copy 2015 Hamid Reza Moghimi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Cellular uptake and cytoplasmic release of liposomal antisense oligonucleotides (AsODNs) which can act as rate-limiting stepsare still remained to be completely optimized Here the possibility of enhancing such processes at cellular and animal levels bycineole as a penetration enhancer was investigated A cationic nanoliposome containing an AsODN against PKC-120572 and a cineole-containing nanoliposomewere prepared and characterizedThe effect of nanoliposomal cineole on sequence-specific cytotoxicity ofnanoliposomal AsODN against A549 was studied in vitro (MTT flow cytometry fluorescence microscopy and real time PCR) andin vivo (xenograft lung tumor in nudemice) using different concentrations and treatment times Results showed specific cytotoxicityof nanoliposomal AsODN was increased significantly from 11 to 25 when A549 cells were exposed to 10 120583gmL cineole for 1 or4 hours This inhibitory effect was further increased to about 40 when the concentration was increased to 40120583gmL for 1 hourIn animal studies cineole significantly decreased the tumor volume (about 75) and increased its doubling time from 13 days to 31days A linear relationship exists between cineole concentration and its enhancement effects Finally it was concluded that cineoleand possibly other membrane fluidizers can improve nanoliposomal gene therapy at cellular and animal levels

1 Introduction

Antisense oligonucleotides (AsODNs) targeting specificgene or mRNA are capable of potently downregulatingproliferation and invasion in human cancer cellsTheymainlyact through RNAse-H activation or hybrid arrest (ie stericblockage of translation) [1 2] Being polyanionic and largeAsODNs practically are unable to permeate cell membraneswell and therefore they have compromised biological activ-ity Rationally designed carriers can help to overcome theserestrictions [3]

Cationic nanoliposomes are among the most widelyused nonviral carriers for macromolecules They encapsulatenucleic acids efficiently through electrostatic interaction andcondense them to form complexes called lipoplexes [4]Liposomal encapsulation of AsODNs improves their trans-fection efficiency and increases their resistance to nucleasedegradation [5 6] These carriers are mainly internalizedinto the target cells through endocytosis [7ndash9] AfterwardAsODNs have to be released from endosomal compartmentinto cytoplasm throughmembrane destabilization and fusionmechanisms However lysosomal degradation of AsODNs

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2015 Article ID 967473 10 pageshttpdxdoiorg1011552015967473

2 Journal of Nanomaterials

can occur especially if AsODNs fail to be released from endo-somes at early stages as pH falls in endosomal compartmentlater [10 11] After release AsODNs may act in the cytoplasmor may migrate from cytoplasm into nucleus [12]

Among the above-mentioned steps cellular uptake andrelease from endosomes are the most significant barriersin antisense therapy [13 14] To overcome these barriersdifferent strategies have been employed such as incorporat-ing nonbilayer forming lipids (eg DOPE) into liposomesapplication of cell penetrating peptides (CPPs) [15ndash19] oremployment of physicalmethods such as ultrasound [20] thatsomehow are difficult to be employed However despite allof these efforts the problem has remained to be completelysolved by a safe easy and economic method

On the other hand there are several molecules calledpenetration enhancers that are widely used to diminishthe barrier function of different biological membranes (egstratum corneum in skin) to facilitate drug delivery to andthrough the tissue [21] Some of these molecules work bydisruption of lipid bilayers which are also present in theliposomes cell membrane and endosomes Therefore it wasdecided here to investigate the application of suchmembranefluidizing agents (penetration enhancers) as a novel strategyfor improving delivery of liposomal ODN in both in vitro(cell lines) and in vivo (animal) models Among differentchemical penetration enhancers is cineole which acts mainlythrough lipid bilayer fluidization and its mechanism of actionis well documented [22ndash24] and was chosen for the presentinvestigation

Cineole the major component of eucalyptus oil belongsto one of the biggest class of skin penetration enhancers(ie terpenes) and has been shown to increase permeationof hydrophilic and lipophilic drugs through human skin [225] lamellar model structures [22 26] and rat skin [27]Its mechanisms of action are said to be extraction of lipidsand keratin denaturation [28] increase in fluidity of lamellarmembranes [22 29] and improving drug partitioning to coreof lamellar membranes [23 30] most of which are expectedto act on liposomes

In this investigation based on earlier survey PKC-120572 as animpressive inhibitory target for gene therapy in adenocarci-noma cells was selectedThe impact of cineole on efficiency ofliposomal AsODNs against protein kinase C-120572 (PKC-120572) wasevaluated through studying molecular transport and viabilityof human non-small-cell lung adenocarcinoma (A549) cellsand suppression of tumor growth in A549 xenograftmodel innude mice [31]

2 Experimental Section

21 Materials DOTAP PEG2000-DSPE egg PC wereobtained from Lipoid GMBH (Ludwigshafen Germany)DSPC was purchased from Northern Lipids (VancouverCanada) Cholesterol (Chol) was purchased from SigmaChemical Company (St Louis MO) A 20-mer phosphorot-hioate oligodeoxynucleotide 51015840-TsCsCs AsTsGs AsCsGsAsAsGs TsAsCs AsGsCs CsGs-31015840 (AsODN) directed againstPKC-120572 mRNA and its random or scrambled sequence(ScODN) 51015840-CsGsAs GsCsAs CsGsCs AsGsTs AsTsCs

AsCsTs AsGs-31015840 that was used as a control oligonucleotide(ScODN) were synthesized by Bioneer (Korea) [31]Fluorescein isothiocyanate tagged phosphorothioateoligonucleotide (FITC-ON) was synthesized and purifiedby Synthegen (Houston TX USA) Fetal bovine serumDulbeccorsquos modified Eaglersquos medium RPMI 1640 mediumtrypan blue stain and trypsin-EDTA solution werepurchased from Gibco BRL 18 cineole (gt99) andMTT were obtained from Sigma Chemical Company (StLouis MO) All other used reagents were of analytical grade

22 Methods

221 Preparation and Characterization of Cationic LiposomesPEG stabilized ODN-encapsulated liposomes (also calledstabilized antisense lipid particles or SALPs) were preparedby the method described previously [6 32] Briefly ethanoliclipid solution was injected into ODN solution (10 120583M) incitrate buffer (pH = 4) Lipid mixture was composed ofDSPCCholPEG lipidDOTAP (20 45 10 25 mol ) Dial-ysis was performed against citrate buffer for 2 hours and sub-sequently HEPES buffered saline (HBS pH = 74) overnightDEAE-Sepharose CL-6B gel chromatography column wasprepared according to the instructions of the manufacturerand unencapsulated ODN was removed using this anion-exchanger resin The nanoliposomes were then stored at 4∘Cuntil use within one month

Particle size and zeta-potential of SALPswere determinedby Malvern Zetasizer (UK) ODN content was measuredby spectrophotometry (Shimadzu Japan) after solubilizingnanoliposomes [32] and phospholipid content was deter-mined after Bligh and Dyer lipid extraction [33] by Stewartmethods [34] (three replicates each) Nanoliposomal encap-sulation efficiency at any step was then calculated using

EE = [(C2L2)(C1L1)

] times 100 (1)

where C2 and L2 are ODN and lipid content of nanolipo-somes at any given stage respectively and C1 and L1 arerespectively initial ODN and lipid content of nanoliposomes

222 Preparation of Liposomal Cineole by Thin Layer FilmHydration Cineole is a lipophilic compound and it is notpossible to deliver it by simple aqueous solution For in vitroapplication of such compounds DMSO or other organicsolvents are usually used to solve material in culture mediaFor in vivo purpose (especially in our survey) however it isbetter to avoid organic solvents due to their cytocidal effectand interference in experimental results [35] As advantagesof liposome as vehicle for essential oils have been shown [36]it was decided here to formulate cineole as nanoliposomes

Cineole-containing nanoliposomeswere prepared by thinfilm hydration method with modification of Sinico method[36] Briefly egg phosphatidylcholine (egg PC)DOTAPcholcineole (14 1 7 78mol) were dissolved in 10mL chloro-form and the solvent was evaporated in a rotary balloon andshaked for 2 hours at 40∘C The obtained lipid film was thenhydrated with HBS (pH 74) to form initial nanoliposomes at40∘C Nanoliposomes were then extruded (3 times through

Journal of Nanomaterials 3

200 nm and 3 times through 100 nm) polycarbonate filtermembranes (Millipore USA) and subsequently purified byG50 Sephadex column to separate free cineole

223 Cineole Assay and Encapsulation Efficiency Cineole-containing nanoliposomeswere destroyed in an equal volumeof methanol to give a clear solution and the solution wasthen assayed for cineole by a GC method using a Shimadzugas chromatography system using BPX1 column and con-nected to FID detector (Shimadzu Co Japan) This methodemployed a calibration curve in the range of 003 to 10mgmLusing solution of cineole in watermethanol (50 50) 119899 = 3The encapsulation efficiency (EE ) of liposomal cineole wascalculated by (1)

224 Cell Culture Studies A549 cells obtained from PasteurInstitute (Tehran Iran) were grown in RPMI 1640 mediumsupplemented with 10 heat-inactivated FBS 100UmLpenicillin and 100 120583gmL streptomycin They were main-tained at 37∘C in a 5 CO

2-incubator (Heraeus Germany)

Cell viability was evaluated either by cell count using trypanblue stain 04 under inverted microscope (Leica Germany)or by MTT assay [37]

To determine enhancer dose the cytotoxic effect ofliposomal cineole on cells was first evaluated at 1 to 40120583gmLcineole concentration and incubation time of 1 and 4 hoursfollowed by cell viability evaluation after 48 hours by MTTassay

For enhancement studies the cells were first treated for1 or 4 hours with nanoliposomal cineole at 1ndash10 120583gmL or1ndash40120583gmL concentration (in nontoxic ranges) The super-natants of cells were then removed and cells were treatedwith nanoliposomal preparations containing either AsODNor ScODN at 150 nM ODN in the culture medium for 48hours After this period sequence specific-antisense activityof nanoliposomal ODNs was evaluated by MTT The sameexperiments were performed in the absence of cineole ascontrol

225 Flow Cytometry Quantitative cellular uptake of lipo-somes was evaluated by flow cytometry 300000 cells wereseeded in each well of 6-well plates (NUNC Denmark) After24-hour incubation the cells were transfected for 4 hoursby 300 nM FITC-ODN containing liposomes either at 4∘Cor at 37∘C Cells were cotreated in some wells with sodiumazide (10mM) as active transport inhibitor and in some wellscells were treated with liposomal cineole (1) Cells weredetached from the plates suspended in culture mediumand then centrifuged Cell pellets were then reconstitutedin 500 120583L of either cold PBS or monensin solution (20120583M)in PBS Cells were kept cold (on ice) until the measure-ment of the cell-associated fluorescence by FACSCaliburdual laser flow cytometer (Becton Dickinson USA) CellQuest software (Becton Dickinson USA) was used for dataacquisition through FL1 (530 nm) and analysis for 10000gated events Cell-associated fluorescence intensities wereexpressed as the average of these measurements plusmn SE(119899 = 3)

226 Fluorescence Microscopy Transfected cells were fixedwith paraformaldehyde and then were permeabilized withTriton X-100 (04) Subsequently the nucleus was stainedwith DAPI (50120583M) Cellular internalization of liposomalFITC-ODNwas studied byAxiovert fluorescencemicroscope(Zeiss Germany) using AxioVision software (Zeiss Ger-many)

227 RT-PCR Assay of Target Gene Expression Cells wereseeded in 6-well plates one day prior to transfection TotalRNA of 6 hours treated cells (with ODNs alone or ODNs +liposomal cineol) was extracted with TRIzol reagent accord-ing to the manufacturerrsquos instructions RNA concentrationand purity were determined by UV-spectrophotometry at260 nm RNA quality was studied by 1 agarose gel elec-trophoresis followed by ethidium bromide staining Prior toelectrophoresis RNA was treated with DNase and purifiedwith Qiagen RNAeasy mini column One microgram of totalRNAwas first reverse-transcribedwith 2 120583L randomhexamerto cDNA and then was applied for PCR amplification [30]PCR amplification was run for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as house-keeping gene in PCRthermal cycler (MJ Research MD) [31] A volume of 10 120583LPCR reaction for PKC-120572 along with its respective humanGAPDHwas loaded into 1 agarose gel in Tris-borate EDTA(TBE) buffer 05x and amplified segments were detected byethidium bromide staining Data were analyzed using UN-SCAN-IT software (Silk Scientific Inc) The experiment wasperformed in triplicate

23 In Vivo Studies

231 Tumor Induction in Nude Mice Female Balbc nunumice (6 weeks old weighing 18 plusmn 2 g) were purchased fromResearch Center for Science and Technology in Medicine inTehran University of Medical Sciences (Tehran Iran) andmaintained at an animal facility in a light- and temperature-controlled aseptic environment The handling of mice andexperimental procedures were conducted in accordance withthe protocols of Ethics Committee of Shaheed BeheshtiUniversity of Medical Sciences

8 times 106 A549 cells were harvested and resuspendedin 200120583L of RPMI 1640 media The cell suspension wasinjected into the right flank of the mice subcutaneously toinduce the initial tumor [38] Mice were then monitoredand checked for tumoral volume every other day until thetumor volumes reached the suitable size of approximately500mm3 Tumor size was measured using a caliper acrossits two perpendicular diameters [39]The initial tumors werethen removed cut into pieces and then passaged into othermice by implanting tumor pieces into the right flank of everyanimal as secondary tumors (xenograft tumors) The growthcurve of the tumors and doubling time of their volumewere then evaluated using Skipper model [40] When thesecondary tumor volumes reached a mean value of around100mm3 the time was considered as time-zero and the micewere randomly assigned to groups of 3 mice for furtherstudies and treatments as described later


Table 1 Properties of prepared oligodeoxy nucleotide- (ODN-) and cineole-containing nanoliposomes

Formulation Size (nm) Zeta potential (mV) Encapsulation efficiency ()ODN-containing liposomes 115 plusmn 50 08 plusmn 07 875 plusmn 38Cineole-containing liposomes 128 plusmn 27 68 plusmn 06 677 plusmn 03

232 Animal Studies of Antisenses ODNs and EnhancementEffect of Liposomal Cineole The antitumoral effects of lipo-somal antisenses and the enhancement effects of cineoleon this process were investigated in tumor-bearing miceNanoliposomal formulation was applied intratumorally at05mgODNkg using approximately 100 120583L of nanoliposo-mal suspension containing 100 120583gmL antisense for eachmouse Cineole-containing nanoliposome was used as pre-treatment (pretreatment injection was selected based onour finding in lab and comparison with cotreatment) byapplying 100 120583L of nanoliposomal cineole (80 120583gmL) asintratumoral injection 2 hours prior to nanoliposomal ODNapplication This process (application of cineole and ODNnanoliposomes) was performed every 4 days for 12 daysVolumes of tumors were measured 2-3 times a week fromtime zero to 1 month after the first injection

233 Hematoxylin and Eosin Staining At the end of theobservation period mice were sacrificed and tumors wereremoved cut and fixed in 10 formalin prior to paraffin pro-cessing and embedding for further pathological studies Rou-tine hematoxylin and eosin (HE) staining [41] was carriedout on harvested tumor samples Slides were then observedunder light microscope and the images were captured andanalyzed for cell death pattern Based on morphology ofcell and nucleus membranes and their content appearance aportion of dead cells (necrotic or apoptotic cells) in each slidewere measured

24 Statistics Data are presented as mean plusmn standard errorfor 3 replicates unless indicated differently Linear regressionand curve fitting were performed by Graphpad Prism soft-ware (San Diego CA) Statistical comparisons were madeusing SPSS statistical software (version 180) through two-sided ANOVA and Tukeyrsquos post hoc testThe differences wereconsidered significant when 119875 lt 005

3 Results

31 Characterization of Liposomes Prepared ODN-contain-ing liposomes showed a nearly neutral zeta potential (08mV)and size of about 115 nm (Table 1)These values did not changesignificantly during 6 months of storage at 4∘C (119875 gt 005)The encapsulation efficiency of ODN-containing liposomeswas calculated to be more than 87

Cineole-containing liposomes showed size and encap-sulation efficiency of about 130 nm and 68 respectively(Table 1) These particles showed a mild positive charge(about 7mV Table 1) that is expected to reduce their aggre-gation and might improve their efficacy [41]

The size of both liposomes used in the present study (115and 130 nm) is close to the optimum size for liposomal drugdelivery (around 100 nm) [42]

120

100

80

60

40

00 05 10 15 20

Viab

ility

()

Log cineole concentration (120583gmL)

4-hour pretreatment1-hour pretreatment

Figure 1 Effect of concentration and exposure time on cytotoxicityof nanoliposomal cineole against A549 cells in vitro Data arepresented as mean plusmn standard

32 Determination of Cineole Cytotoxicity and EnhancerConcentration Figure 1 shows the effect of liposomal cineoleon the viability of A549 cells in vitro As is shown cineoledid not affect cell viability for 1-hour pretreatment overthe whole concentration range used here (1 to 40120583gmL)However after 4-hour pretreatment although there is nosignificant cytotoxicity below 10120583gmL the viability of cellsdecreased significantly at higher concentrations of 20 and40 120583gmL Segmental analysis showed that a linear rela-tionship exists between cell viability and log concentration(Figure 1) The slope of the corresponding line was definedhere as antiproliferative-concentration sensitivity (ACS) thatwas minus60 plusmn 55 in the range of 10ndash40 120583gmL for 4-hourpretreatmentThe slope (ACS) for 1-hour pretreatment (19plusmn05) and 4-hour pretreatment below 10 120583gmL (minus56plusmn31) wasnot significantly different from zero (119875 gt 005) Therefore allsubsequent cineole-treatment protocols employed nontoxiccondition based on the above results

33 Effect of Cineole on Liposomal ODN Cytotoxicity In VitroFigure 2 provides the effects of different cineole concentra-tions and treatment protocols on antiproliferative effects ofliposomal ODNs Results showed that control nanoliposomalODN (ScODN) does not show a significant cytotoxicity inthe absence or presence of 1-hour cineole pretreatment over1ndash40 120583gmL The relationship between cell viability and logconcentration was found to be linear with a slope (ACS) of(minus08 plusmn 07) that is not different from zero (119875 gt 005) Whenthe pretreatment time was increased to 4 hours the nanoli-posomal ScODN showed a slight decrease in the cell viability


00 05 10 15 2040

60

80

100

120

1-hour cineole + AsODN1-hour cineole + ScODN4-hour cineole + AsODN4-hour cineole + ScODN

Viab

ility

()


Figure 2 Effect of cineole pretreatment on antiproliferative actionof nanoliposomal antisense oligonucleotide (AsODN) in compari-son to its control (ScODN) at different treatment times Data arepresented as mean plusmn standard error (119899 = 6)

upon cineole concentration increase (slope = minus47 plusmn 02) andthe viability reached to a value of about 91 at 10 120583gmLThese data were used to select nontoxic concentration ofcineole to apply in gene delivery experiments

Nanoliposomal antisense (AsODN) showed a 20 reduc-tion in cell viability in the absence of cineole that was sig-nificant in comparison to ScODN Cineole treatment causeda concentration-dependent increase in antisense effect forboth 1- and 4-hour pretreatment times (Figure 2) The cellviability reached a value of about 70 at 10 gmL and 55at 40 120583gmL The slopes of viability versus concentration linewere calculated to be minus157 plusmn 07 and minus181 plusmn 12 for 1- and4-hour cineole pretreatments respectively indicating thatincreased pretreatment time causes a slight increase in thesensitivity

34 Fluorescence Microscopy and Flow Cytometry Figure 3shows that cationic liposomes can increase the cellular uptakeof AsODN very significantly from 90 plusmn 05 in free ODN to avalue of 842 plusmn 39 Cineole was able to further increase thecell-associated fluorescence of liposomal ODN to a value of1343 plusmn 65 (119875 lt 005) In contrast the effect of cineole on thecellular uptake of free ODN was negligible (119875 gt 005)

After decreasing the incubation temperature from 37∘Cto 4∘C cell-associated fluorescence intensity of the SALPsdiminished to 228plusmn52 Almost the same effect was observedwhen the cells were coincubated with sodium azide at 37∘C(intensity of 390 plusmn 43) Cineole treatment did not changethe cellular fluorescence intensity with these inhibitory con-ditions (4∘C or in presence of sodium azide) The fluorescentintensities in the presence of cineol at 4∘C or in the presenceof cineole and sodium azide were calculated to be 223 plusmn 17and 469 plusmn 47 respectively that are comparable to the above-mentioned values Incubation at 4∘C or with sodium azideis expected to block the cellular uptake of the liposomes

With cineolWithout cineol

0 25 50 75 100 125 150

Fluorescence intensity

SALP + Na azide 37∘C

SALP 4∘C

SALP 37∘C

Free AsODN 37∘C

Figure 3 Flow cytometric analysis of the cellular uptake of preparedcationic liposomes (SALPs) containing FITC-labelled antisenseoligodeoxynucleotides (AsODN) in cineole-treated (dark bars) oruntreated (light bars) samples at 37∘C or 4∘C in the presence orabsence of sodium azide treatments Data are presented as mean plusmnstandard error (119899 = 2) Data of untreated samples are fromTamaddon (From [43] with permission from the CorrespondingAuthor and based on the related Copyright Agreements with thepublisher)

These data confirms endocytosis as the main entrance routeof liposomal ODNs

Figure 4 shows that the green fluorescence of FITC-ODNprobe is diffusively scattered in the cells transfected with theODN-containing liposomes (SALPs) (c) while no distinctintracellular fluorescence was observed if the cells incubatedwith free FITC-ODN at the same concentration (a) Cineoletreatment increased the intracellular trafficking of the SALP(d) and the cell-associated fluorescence of FITC-ODN (b)

35 RT-PCR Determination of Antisense Activity SpecificPCR products of 172 and 531 base pairs were obtainedfor PKC-120572 and GAPDH (internal control) respectivelyTreatment with 300 nM AsODN inhibited the gene expres-sion (390 plusmn 71) while no obvious inhibition of PKC-120572 was observed with ScODN Coincubation of the cellswith 1 liposomal cineole at the similar SALP concentrationincreased the sequence-specific inhibition of the target geneby about 2 times to 825 (Figure 5 119875 lt 001)

36 Effect of Cineole on In Vivo Tumor Growth in XenograftModel Tumor growth profile in nude mice (Figure 6(a))showed that nanoliposomal antisense decreases the tumorvolume from 832 to 440mm3 after 30 days Nanoliposomalcineole further increased the antisense activity significantly(119875 lt 005) by decreasing the tumor volume twice more to avalue of 205mm3 Scrambled ODN-containing formulationand liposomal cineole also decreased the tumor volume butthe effect was not that significant (119875 gt 005) (Figure 6)

Data were also interpreted according to tumor volumedoubling time (Figure 6(b)) Tumor volume doubling timein control (nontreated) animals was calculated to be 96 plusmn06 days Nanoliposomal AsODN increased this doublingtime significantly (119875 lt 005) to a value of 134 days Aftercineole treatment the doubling time was further increased(119875 lt 001) more than twofold to 314 days LiposomalScODN (negative control) did not change the doubling


(a)

(b)

(c)

(d)

Figure 4 Epifluorescence microscopy study of FITC-antisense oligodeoxynucleotides (AsODN) cellular distribution Cytoplasmicdistribution of fluorescein green fluorescence (right images) was demonstrated after 4-hour incubation with either free AsODN (a b)liposomal AsODN (c d) cineole-treated free AsODN (b) and cineole-treated liposomal AsODN (d) Left images illustrate DAPI-stainednucleuses of the same systems

time indicating that the increased doubling time of AsODNis related to sequence specific inhibitory effect Liposomalcineole was able to increase the doubling time marginally to123 days

37 Hematoxylin and Eosin Staining Figure 7 illustrates HE-stained images of tumor for different treatmentsThe severity

of cell death was increased upon cineole pretreatment(Figure 7) and more than 84 of cells had died In thegroup that was treated with nanoliposomal AsODN alonevisible areas of necrotic and apoptotic cells can be seenin related slide and almost 324 of cells were dead (Fig-ure 7) Only small punctuate necrosis and apoptosis (ieless than 3) were seen in ScODN-treated group (negative


AsODNScODN

150

125

100

75

50

25

0

Pure cell culture Cineole-containing cell culture

mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)

Figure 5 RT-PCR determination of protein kinase C-120572 (PKC-120572)expression A549 cells were transfected with the liposomal antisense(As) or scrambled (Sc) oligodeoxynucleotides (ODN) Cells wereincubated with either pure cell culture medium alone (left bars) orcineole-containing cell culture medium (right bars) Quantitativeexpressed target mRNA GAPDH data are provided as control

control Figure 7) and no obvious necrosis and apoptosiswere observed in A549 tumors of untreated group (Figure 7)Pattern of cell death and debris of dead cell imply thatapoptosis is probably responsible for tumor inhibition

4 Discussion

Present cell culture studies showed that applied AsODN candecrease cell viability with minor effects from scrambledantisense (control) revealing that the effect of applied anti-sense on cell viability is through specific mRNA inhibitionwhich is in good correlation with Tamaddon findings [43]Animal data also revealed that nanoliposomal antisensewas able to decrease the tumor volume and increase itsdoubling time These findings are in agreement with Yazakiet al who showed that intraperitoneal administration ofphosphorothioate antisense oligonucleotides against PKC-alpha mRNA were able to inhibit xenografted glial tumors innude mice [44] and that of Ying et al who showed tumorinhibition effect of Elk-1 (an antisense ODN against PKC-alpha) in carcinoma both in vitro and in nude mice [45]

Cineole pretreatment increased the antisense effect sig-nificantly by decreasing the tumor volume and increasing itsdoubling time (more than 200) or decreasing cell viabilityin cell culture It has been shown that endocytosis andinteractions of liposomes with cytoplasmic and endosomalmembranes are themainmechanisms for liposomal antisensedelivery [9 43] On the other hand cineole performs itsenhancement effect toward small molecules through eithercomplexation with small polar molecules [23] or lipid bilayerfluidization [22 46] Between these two mechanisms theimportance of complexation might be lower due to themechanism of entrance of these particles into the cell inliposomal delivery Therefore it might be concluded here

that cineole exerts its action through lipid bilayers fluidiza-tion Such a fluidization can increase membrane fusion ordisruption at liposomes cell membrane andor endosomelevels and therefore improve uptake of liposomes into thecell andor increase their endosomal escape This proposedmechanism is in agreement with increased deformability andhigher cutaneous uptake of cineole-containing liposomes incomparison to control liposomes [47] In this direction ithas been shown that presence of 40mol DOPE (a lipidfluidizer) causes enhanced transfection efficiency of Gallipoplex in Hela cells [48] The main action of DOPE isenhancing fusion by producing hexagonal phases thereforeDOPEdecreases the stability of liposomes due to its geometryand it has been shown that in vivo application of DOPE islimited because of its interaction with blood cells [49] andinstability of the liposomes [18] It has also been shown thatpartially substituted polylysine (bilayer destabilizer) causesa dramatic increase by (3ndash45 orders of magnitude) of thetransfection efficiency of DNApolylysine polyplexes [50]

Our results also show that the enhancement effect ofcineole is concentration-dependent which is in good agree-ment with concentration-dependent enhancement effect ofcineole toward permeation of 5-fluorouracil and oestradiolthrough a lamellar liquid crystalline lipid model structure[26] In comparison to urea as penetration enhancer forgene delivery cineol could improve transfection efficiency ofliposomal ODN by around 2-foldmore intensive [51] Resultsalso showed that treatment time affects enhancement effectof cineole but its intensity was found to be less than cineoleconcentration (Figure 2)

The present results also indicate that cell culture datacorrelate well with animal studies in terms of enhancementeffect indicating that cell culture studies might be used forscreening of enhancers against liposomal delivery

Besides enhancement effects cineole showed cytotoxiceffect against A549 cells at high concentrations andor longtreatment times This is in agreement with Bowen and Aliwho showed that some terpenes may show antiproliferativeeffect in cancer cells [52]

Although the data obtained clearly indicates that cineolehas the ability to enhance the cellular delivery of liposomalAsODNs and also improves their antitumor efficacy weassumed that at least the in vitro results should be extended toa wider panel of cell lines to substantiate the conclusion thatcineole increase liposomal AsODNs delivery and antitumoreffects Therefore these steps were also implemented forOV2008 (ovarian carcinoma) and A431 (epidermoid carci-noma) as two control cell lines and results (have not beendemonstrated here) also confirmed the above conclusion

To conclude the present results clearly show that cine-ole and possibly other terpenes and chemical penetrationenhancers are able to increase nanoliposomal AsODN deliv-ery and its antitumoral effect In terms of drug deliveryincreased permeation means either more pharmacologicaleffect or less drug content for the same effect both ofwhich will result in more economic systems for expensivetreatment methods like liposomal gene delivery In thisdirection Dempsey et al have shown that sublethal doses ofchemotherapeutic agents can be combined with membrane


0 5 10 15 20 25 30 35 400

200

400

600

800

1000

Untreated AsODNScODN AsODN + cineoleCineole

Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s

Liposomalcineole liposomal cineole

(b)

Figure 6 Tumor growth profile (a) and doubling time (b) as indicators of inhibitory effects of different antisense (As) or scrambled (Sc)oligodeoxynucleotides (ODN) nanoliposomal formulations in the presence or absence of cineole in nude mice in comparison to untreatedcontrol animal Data are presented as mean plusmn standard error (119899 = 3)

(a) (b)

(c) (d)

Figure 7 Hematoxylineosin staining (parallel to Annexin Kit selection) of untreated tumor (a) and tumor treated by nanoliposomalantisense (b c) or scrambled (d) oligonucleotides in the absence (b d) or presence (c) of cineole

fluidizing treatments (hyperthermia benzyl alcohol andethanol) to produce a significant increase in drug efficacyand apoptosis [53] Other enhancement methods are alsoreported for enhancement of liposomal or nonliposomalODN delivery like sonophoresis [20] electroporation com-plex formation or radiation [54 55] In comparison to suchmethods chemical penetration enhancers are considered lessinvasive readily available easy to apply and less expensive

5 Conclusion

Present investigation clearly shows that cineole and possiblyother membrane fluidizing agents or penetration enhancerscan improve antitumor activity of nanoliposomal antisenseoligonucleotides possibly through improved cellular delivery

and cytoplasmic release Although several other enhance-ment methods are available such chemical enhancementmight provide more versatile less expensive and noninva-sive approaches for gene delivery This strategy can opennew era in the field of liposomal gene therapy Furtherinvestigations including intravenous applicability of cineole-fluidized liposome in animal models and application of otherbiocompatible membrane fluidizing agents are suggested andare in progress in our laboratories

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper


Acknowledgments

This work was supported by Pharmaceutical SciencesResearch Centre at Shahid Beheshti University of MedicalSciences (SBMU) Tehran Iran (Grant no 4008332) toHamid Reza Moghimi This work is from PhD theses ofMostafa Saffari at School of Pharmacy SBMU

References

[1] N Dias and C A Stein ldquoAntisense oligonucleotides basicconcepts and mechanismsrdquoMolecular CancerTherapeutics vol1 no 5 pp 347ndash355 2002

[2] B Yang J Gao Z Rao B Zhang W Ouyang and C YangldquoAntisense oligonucleotide targeting matrix metalloproteinase-7 (MMP-7) changes the ultrastructure of human A549 lungadenocarcinoma cellsrdquo Ultrastructural Pathology vol 35 no 6pp 256ndash259 2011

[3] S Akhtar M D Hughes A Khan et al ldquoThe delivery ofantisense therapeuticsrdquo Advanced Drug Delivery Reviews vol44 no 1 pp 3ndash21 2000

[4] N S Templeton D D Lasic P M Frederik H H Strey DD Roberts and G N Pavlakis ldquoImproved DNA liposomecomplexes for increased systemic delivery and gene expressionrdquoNature Biotechnology vol 15 no 7 pp 647ndash652 1997

[5] F Pastorino D Stuart M Ponzoni and T M Allen ldquoTargeteddelivery of antisense oligonucleotides in cancerrdquo Journal ofControlled Release vol 74 no 1ndash3 pp 69ndash75 2001

[6] S C Semple S KKlimuk TOHarasymet al ldquoEfficient encap-sulation of antisense oligonucleotides in lipid vesicles usingionizable aminolipids formation of novel small multilamellarvesicle structuresrdquo Biochimica et Biophysica Acta vol 1510 no1-2 pp 152ndash166 2001

[7] D S Friend D Papahadjopoulos and R J Debs ldquoEndocytosisand intracellular processing accompanying transfection medi-ated by cationic liposomesrdquo Biochimica et Biophysica Acta vol1278 no 1 pp 41ndash50 1996

[8] Y Xu and F C Szoka Jr ldquoMechanism of DNA release fromcationic liposomeDNA complexes used in cell transfectionrdquoBiochemistry vol 35 no 18 pp 5616ndash5623 1996

[9] G Sahay D Y Alakhova and A V Kabanov ldquoEndocytosis ofnanomedicinesrdquo Journal of Controlled Release vol 145 no 3 pp182ndash195 2010

[10] H Harashima Y Shinohara and H Kiwada ldquoIntracellularcontrol of gene trafficking using liposomes as drug carriersrdquoEuropean Journal of Pharmaceutical Sciences vol 13 no 1 pp85ndash89 2001

[11] J Monkkonen and A Urtti ldquoLipid fusion in oligonucleotideand gene delivery with cationic lipidsrdquo Advanced Drug DeliveryReviews vol 34 no 1 pp 37ndash49 1998

[12] G L Lukacs P Haggie O Seksek D Lechardeur N Freedmanand A S Verkman ldquoSize-dependent DNA mobility in cyto-plasmandnucleusrdquoThe Journal of Biological Chemistry vol 275no 3 pp 1625ndash1629 2000

[13] M B Bally P Harvie F M P Wong S Kong E K Wasan andD L Reimer ldquoBiological barriers to cellular delivery of lipid-based DNA carriersrdquo Advanced Drug Delivery Reviews vol 38no 3 pp 291ndash315 1999

[14] F Shi and D Hoekstra ldquoEffective intracellular delivery ofoligonucleotides in order to make sense of antisenserdquo Journalof Controlled Release vol 97 no 2 pp 189ndash209 2004

[15] M C De Oliveira E Fattal C Ropert C Malvy and PCouvreur ldquoDelivery of antisense oligonucleotides by means ofpH-sensitive liposomesrdquo Journal of Controlled Release vol 48no 2-3 pp 179ndash184 1997

[16] R Rennert C Wespe A G Beck-Sickinger and I NeundorfldquoDeveloping novel hCT derived cell-penetrating peptides withimproved metabolic stabilityrdquo Biochimica et Biophysica Actavol 1758 no 3 pp 347ndash354 2006

[17] I M Hafez S Ansell and P R Cullis ldquoTunable pH-sensitiveliposomes composed ofmixtures of cationic and anionic lipidsrdquoBiophysical Journal vol 79 no 3 pp 1438ndash1446 2000

[18] S W Hui M Langner Y-L Zhao P Ross E Hurley and KChan ldquoThe role of helper lipids in cationic liposome-mediatedgene transferrdquo Biophysical Journal vol 71 no 2 pp 590ndash5991996

[19] L Y Song Q F Ahkong Q Rong et al ldquoCharacteriza-tion of the inhibitory effect of PEG-lipid conjugates on theintracellular delivery of plasmid and antisense DNA mediatedby cationic lipid liposomesrdquo Biochimica et Biophysica ActamdashBiomembranes vol 1558 no 1 pp 1ndash13 2002

[20] Y-K Luo Y-Z Zhao C-T Lu J Tang and X-K Li ldquoAppli-cation of ultrasonic gas-filled liposomes in enhancing transferfor breast cancer-related antisense oligonucleotides an experi-mental studyrdquo Journal of Liposome Research vol 18 no 4 pp341ndash351 2008

[21] A C Williams H G M Edwards E E Lawson and BW Barry ldquoMolecular interactions between the penetrationenhancer 18-cineole and human skinrdquo Journal of RamanSpectroscopy vol 37 no 1ndash3 pp 361ndash366 2006

[22] H R Moghimi A C Williams and B W Barry ldquoA lamellarmatrixmodel for stratumcorneum intercellular lipids V Effectsof terpene penetration enhancers on the structure and thermalbehaviour of the matrixrdquo International Journal of Pharmaceu-tics vol 146 no 1 pp 41ndash54 1997

[23] H RMoghimi A CWilliams and BW Barry ldquoEnhancementby terpenes of 5-fluorouracil permeation through the stratumcorneum model solvent approachrdquo Journal of Pharmacy andPharmacology vol 50 no 9 pp 955ndash964 1998

[24] H R Moghimi A C Williams and B W Barry ldquoA lamellarmatrix model for stratum corneum intercellular lipids IVEffects of terpene penetration enhancers on the permeation of5-fluorouracil and oestradiol through the matrixrdquo InternationalJournal of Pharmaceutics vol 145 no 1-2 pp 49ndash59 1996

[25] A C Williams and B W Barry ldquoPenetration enhancersrdquoAdvanced Drug Delivery Reviews vol 56 no 5 pp 603ndash6182004


[27] Z S Saify O Ahsan and A Dayo ldquoCineole as skin penetrationenhancerrdquo Pakistan Journal of Pharmaceutical Sciences vol 13no 1 pp 29ndash32 2000

[28] A Ahad M Aqil K Kohli Y Sultana M Mujeeb and A AlildquoInteractions between novel terpenes and main components ofrat and human skin mechanistic view for transdermal deliveryof propranolol hydrochloriderdquoCurrent DrugDelivery vol 8 no2 pp 213ndash224 2011

[29] J L Anjos D de Sousa Neto and A Alonso ldquoEffects of 18-cineole on the dynamics of lipids and proteins of stratum


corneumrdquo International Journal of Pharmaceutics vol 345 no1-2 pp 81ndash87 2007

[30] C M Heard D Kung and C P Thomas ldquoSkin penetrationenhancement of mefenamic acid by ethanol and 18-cineolecan be explained by the lsquopullrsquo effectrdquo International Journal ofPharmaceutics vol 321 no 1-2 pp 167ndash170 2006

[31] H-F Song Z-M Tang S-J Yuan B-Z Zhu and X-W LiuldquoAntisense candidates against protein kinase C-120572 designedbased on phylogenesis and simulant structure of mRNArdquo ActaPharmacologica Sinica vol 24 no 3 pp 269ndash276 2003

[32] NMaurer K FWongH Stark et al ldquoSpontaneous entrapmentof polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomesrdquoBiophysical Journal vol 80 no5 pp 2310ndash2326 2001

[33] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959

[34] J C M Stewart ldquoColorimetric determination of phospholipidswith ammonium ferrothiocyanaterdquo Analytical Biochemistryvol 104 no 1 pp 10ndash14 1980

[35] W Wolfgang and S S Ulrike Eds Gene Therapy of CancerMethods and Protocols vol 542 ofMethods inMolecular BiologyHumana Press New York NY USA 2nd edition 2009

[36] C Sinico A De Logu F Lai et al ldquoLiposomal incorporation ofArtemisia arborescens L essential oil and in vitro antiviral activ-ityrdquo European Journal of Pharmaceutics and Biopharmaceuticsvol 59 no 1 pp 161ndash168 2005

[37] T Mosmann ldquoRapid colorimetric assay for cellular growth andsurvival application to proliferation and cytotoxicity assaysrdquoJournal of Immunological Methods vol 65 no 1-2 pp 55ndash631983

[38] S Hou Z Zhao F Yan et al ldquoGenetic transfer of PNAS-4induces apoptosis and enhances sensitivity to gemcitabine inlung cancerrdquo Cell Biology International vol 33 no 3 pp 276ndash282 2009

[39] I-Y Kim Y-S Kang D S Lee et al ldquoAntitumor activity ofEGFR targeted pH-sensitive immunoliposomes encapsulatinggemcitabine inA549 xenograftnudemicerdquo Journal of ControlledRelease vol 140 no 1 pp 55ndash60 2009

[40] E J Freireich E A Gehan D P Rall L H Schmidt and HE Skipper ldquoQuantitative comparison of toxicity of anticanceragents in mouse rat hamster dog monkey and manrdquo CancerChemotherapy Reports vol 50 no 4 pp 219ndash244 1966

[41] R Zhao W Yang Z Wang G Li W Qin and J WangldquoTreatment of transplanted tumor of lung adenocarcinomaA549 transfected by human somatostatin receptor subtype 2(hsstr2) genewith 188Re-RC-160rdquoNuclearMedicine and Biologyvol 37 no 8 pp 977ndash987 2010

[42] D C Drummond O Meyer K Hong D B Kirpotin andD Papahadjopoulos ldquoOptimizing liposomes for delivery ofchemotherapeutic agents to solid tumorsrdquo PharmacologicalReviews vol 51 no 4 pp 691ndash743 1999

[43] A M Tamaddon Effect of some lipid bilayer-destabilizers onrelease of antisense oligodeoxynucleotides from cationic liposomesand their cytotoxicity [PhD thesis] Shahid Beheshti Universityof Medical Sciences Tehran Iran 2006

[44] T Yazaki S Ahmad A Chahlavi et al ldquoTreatment of glioblas-toma U-87 by systemic administration of an antisense proteinkinase C-120572 phosphorothioate oligodeoxynucleotiderdquoMolecularPharmacology vol 50 no 2 pp 236ndash242 1996

[45] T-H Ying Y-H Hsieh Y-S Hsieh and J-Y Liu ldquoAntisenseoligonucleotide Elk-1 suppresses the tumorigenicity of humanhepatocellular carcinoma cellsrdquo Cell Biology International vol32 no 2 pp 210ndash216 2008

[46] P A Cornwell B W Barry J A Bouwstra and G SGooris ldquoModes of action of terpene penetration enhancersin human skin differential scanning calorimetry small-angleX-ray diffraction and enhancer uptake studiesrdquo InternationalJournal of Pharmaceutics vol 127 no 1 pp 9ndash26 1996

[47] S Mura M Manconi C Sinico D Valenti and A M FaddaldquoPenetration enhancer-containing vesicles (PEVs) as carriersfor cutaneous delivery of minoxidilrdquo International Journal ofPharmaceutics vol 380 no 1-2 pp 72ndash79 2009

[48] MD Kearns Y N Patel andM Savva ldquoPhysicochemical char-acteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPErdquo Chem-istry and Physics of Lipids vol 163 no 8 pp 755ndash764 2010

[49] F Sakurai T Nishioka H Saito et al ldquoInteraction betweenDNA-cationic liposome complexes and erythrocytes is animportant factor in systemic gene transfer via the intravenousroute inmice the role of the neutral helper lipidrdquoGeneTherapyvol 8 no 9 pp 677ndash686 2001

[50] P Midoux C Pichon J-J Yaouanc and P-A Jaffres ldquoChem-ical vectors for gene delivery a current review on polymerspeptides and lipids containing histidine or imidazole as nucleicacids carriersrdquo British Journal of Pharmacology vol 157 no 2pp 166ndash178 2009

[51] M Saffari A M Tamaddon F H Shirazi M A Oghabian andH R Moghimi ldquoImproving cellular uptake and in vivo tumorsuppression efficacy of liposomal oligonucleotides by urea as achemical penetration enhancerrdquo Journal of Gene Medicine vol15 no 1 pp 12ndash19 2013

[52] I D Bowen and Y A Ali ldquoAnti-Tumour Terpene CompoundsrdquoUnited States Patents 20070259056 2007

[53] N C Dempsey H E Ireland C M Smith C F Hoyle andJ H H Williams ldquoHeat Shock Protein translocation inducedby membrane fluidization increases tumor-cell sensitivity tochemotherapeutic drugsrdquo Cancer Letters vol 296 no 2 pp257ndash267 2010

[54] D G Spiller R V Giles J Grzybowski D M Tidd and R EClark ldquoImproving the intracellular delivery and molecular effi-cacy of antisense oligonucleotides in chronic myeloid leukemiacells a comparison of streptolysin-O permeabilization electro-poration and lipophilic conjugationrdquo Blood vol 91 no 12 pp4738ndash4746 1998

[55] R A Abela J Qian L Xu T S Lawrence and M ZhangldquoRadiation improves gene delivery by a novel transferrin-lipoplex nanoparticle selectively in cancer cellsrdquo Cancer GeneTherapy vol 15 no 8 pp 496ndash507 2008

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



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CompositesJournal of

NanoparticlesJournal of



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Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


can occur especially if AsODNs fail to be released from endo-somes at early stages as pH falls in endosomal compartmentlater [10 11] After release AsODNs may act in the cytoplasmor may migrate from cytoplasm into nucleus [12]

Among the above-mentioned steps cellular uptake andrelease from endosomes are the most significant barriersin antisense therapy [13 14] To overcome these barriersdifferent strategies have been employed such as incorporat-ing nonbilayer forming lipids (eg DOPE) into liposomesapplication of cell penetrating peptides (CPPs) [15ndash19] oremployment of physicalmethods such as ultrasound [20] thatsomehow are difficult to be employed However despite allof these efforts the problem has remained to be completelysolved by a safe easy and economic method

On the other hand there are several molecules calledpenetration enhancers that are widely used to diminishthe barrier function of different biological membranes (egstratum corneum in skin) to facilitate drug delivery to andthrough the tissue [21] Some of these molecules work bydisruption of lipid bilayers which are also present in theliposomes cell membrane and endosomes Therefore it wasdecided here to investigate the application of suchmembranefluidizing agents (penetration enhancers) as a novel strategyfor improving delivery of liposomal ODN in both in vitro(cell lines) and in vivo (animal) models Among differentchemical penetration enhancers is cineole which acts mainlythrough lipid bilayer fluidization and its mechanism of actionis well documented [22ndash24] and was chosen for the presentinvestigation

Cineole the major component of eucalyptus oil belongsto one of the biggest class of skin penetration enhancers(ie terpenes) and has been shown to increase permeationof hydrophilic and lipophilic drugs through human skin [225] lamellar model structures [22 26] and rat skin [27]Its mechanisms of action are said to be extraction of lipidsand keratin denaturation [28] increase in fluidity of lamellarmembranes [22 29] and improving drug partitioning to coreof lamellar membranes [23 30] most of which are expectedto act on liposomes

In this investigation based on earlier survey PKC-120572 as animpressive inhibitory target for gene therapy in adenocarci-noma cells was selectedThe impact of cineole on efficiency ofliposomal AsODNs against protein kinase C-120572 (PKC-120572) wasevaluated through studying molecular transport and viabilityof human non-small-cell lung adenocarcinoma (A549) cellsand suppression of tumor growth in A549 xenograftmodel innude mice [31]

2 Experimental Section

21 Materials DOTAP PEG2000-DSPE egg PC wereobtained from Lipoid GMBH (Ludwigshafen Germany)DSPC was purchased from Northern Lipids (VancouverCanada) Cholesterol (Chol) was purchased from SigmaChemical Company (St Louis MO) A 20-mer phosphorot-hioate oligodeoxynucleotide 51015840-TsCsCs AsTsGs AsCsGsAsAsGs TsAsCs AsGsCs CsGs-31015840 (AsODN) directed againstPKC-120572 mRNA and its random or scrambled sequence(ScODN) 51015840-CsGsAs GsCsAs CsGsCs AsGsTs AsTsCs

AsCsTs AsGs-31015840 that was used as a control oligonucleotide(ScODN) were synthesized by Bioneer (Korea) [31]Fluorescein isothiocyanate tagged phosphorothioateoligonucleotide (FITC-ON) was synthesized and purifiedby Synthegen (Houston TX USA) Fetal bovine serumDulbeccorsquos modified Eaglersquos medium RPMI 1640 mediumtrypan blue stain and trypsin-EDTA solution werepurchased from Gibco BRL 18 cineole (gt99) andMTT were obtained from Sigma Chemical Company (StLouis MO) All other used reagents were of analytical grade

22 Methods

221 Preparation and Characterization of Cationic LiposomesPEG stabilized ODN-encapsulated liposomes (also calledstabilized antisense lipid particles or SALPs) were preparedby the method described previously [6 32] Briefly ethanoliclipid solution was injected into ODN solution (10 120583M) incitrate buffer (pH = 4) Lipid mixture was composed ofDSPCCholPEG lipidDOTAP (20 45 10 25 mol ) Dial-ysis was performed against citrate buffer for 2 hours and sub-sequently HEPES buffered saline (HBS pH = 74) overnightDEAE-Sepharose CL-6B gel chromatography column wasprepared according to the instructions of the manufacturerand unencapsulated ODN was removed using this anion-exchanger resin The nanoliposomes were then stored at 4∘Cuntil use within one month

Particle size and zeta-potential of SALPswere determinedby Malvern Zetasizer (UK) ODN content was measuredby spectrophotometry (Shimadzu Japan) after solubilizingnanoliposomes [32] and phospholipid content was deter-mined after Bligh and Dyer lipid extraction [33] by Stewartmethods [34] (three replicates each) Nanoliposomal encap-sulation efficiency at any step was then calculated using

EE = [(C2L2)(C1L1)

] times 100 (1)

where C2 and L2 are ODN and lipid content of nanolipo-somes at any given stage respectively and C1 and L1 arerespectively initial ODN and lipid content of nanoliposomes

222 Preparation of Liposomal Cineole by Thin Layer FilmHydration Cineole is a lipophilic compound and it is notpossible to deliver it by simple aqueous solution For in vitroapplication of such compounds DMSO or other organicsolvents are usually used to solve material in culture mediaFor in vivo purpose (especially in our survey) however it isbetter to avoid organic solvents due to their cytocidal effectand interference in experimental results [35] As advantagesof liposome as vehicle for essential oils have been shown [36]it was decided here to formulate cineole as nanoliposomes

Cineole-containing nanoliposomeswere prepared by thinfilm hydration method with modification of Sinico method[36] Briefly egg phosphatidylcholine (egg PC)DOTAPcholcineole (14 1 7 78mol) were dissolved in 10mL chloro-form and the solvent was evaporated in a rotary balloon andshaked for 2 hours at 40∘C The obtained lipid film was thenhydrated with HBS (pH 74) to form initial nanoliposomes at40∘C Nanoliposomes were then extruded (3 times through












23 In Vivo Studies









3 Results




120

100

80

60

40

00 05 10 15 20

Viab

ility

()







00 05 10 15 2040

60

80

100

120


Viab

ility

()








0 25 50 75 100 125 150



SALP 4∘C

SALP 37∘C

Free AsODN 37∘C








(a)

(b)

(c)

(d)






AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials














23 In Vivo Studies









3 Results




120

100

80

60

40

00 05 10 15 20

Viab

ility

()







00 05 10 15 2040

60

80

100

120


Viab

ility

()








0 25 50 75 100 125 150



SALP 4∘C

SALP 37∘C

Free AsODN 37∘C








(a)

(b)

(c)

(d)






AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









3 Results




120

100

80

60

40

00 05 10 15 20

Viab

ility

()







00 05 10 15 2040

60

80

100

120


Viab

ility

()








0 25 50 75 100 125 150



SALP 4∘C

SALP 37∘C

Free AsODN 37∘C








(a)

(b)

(c)

(d)






AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




00 05 10 15 2040

60

80

100

120


Viab

ility

()








0 25 50 75 100 125 150



SALP 4∘C

SALP 37∘C

Free AsODN 37∘C








(a)

(b)

(c)

(d)






AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a)

(b)

(c)

(d)






AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




AsODNScODN

150

125

100

75

50

25

0


mRN

A (n

orm

aliz

ed b

y G

APD

H) (

)



4 Discussion










0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 5 10 15 20 25 30 35 400

200

400

600

800

1000


Trea

tmen

t ini

tiatio

n

Days

Volu

me o

f tum

or (m

m3)

(a)

Untreated

Untreated

ScODN

ScODN

Cineole

AsODN

AsODN

AsODN + cineole

AsODN +

40

30

20

10

0

Day

s


(b)


(a) (b)

(c) (d)



5 Conclusion






Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Acknowledgments


References

































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Research Article In Vitro and In Vivo Enhancement of ...downloads.hindawi.com/journals/jnm/2015/967473.pdf · Islamic Azad University (IAUPS), Tehran, Iran Pharma Chemie Company,