Cationic polyene phospholipids as DNA carriers for ocular gene therapy

Research ArticleCationic Polyene Phospholipids as DNACarriers for Ocular Gene Therapy

Susana Machado1 Sofia Calado12 Diogo Bitoque13 Ana Vanessa Oliveira12

Christer L Oslashpstad4 Muhammad Zeeshan5 Hans-Richard Sliwka4 Vassilia Partali4

Michael D Pungente5 and Gabriela A Silva16

1 Center for Biomedicine and Structural Biology (CBME) Institute for Biotechnology and Bioengineering (IBB)University of Algarve 8005-139 Faro Portugal

2 Doctoral Program in Biomedical Sciences Department of Biomedical Sciences and MedicineUniversity of Algarve 8005-139 Faro Portugal

3 ProRegeM Doctoral Program Department of Biomedical Sciences and Medicine University of Algarve 8005-139 Faro Portugal4Department of Chemistry Norwegian University of Science and Technology 7491 Trondheim Norway5 Premedical Unit Weill Cornell Medical College in Qatar PO Box 24144 Doha Qatar6Department of Biomedical Sciences and Medicine University of Algarve 8005-139 Faro Portugal

Correspondence should be addressed to Michael D Pungente mdp2001qatar-medcornelleduand Gabriela A Silva gasilvaualgpt

Received 28 April 2014 Accepted 18 June 2014 Published 24 July 2014

Academic Editor Taiyoun Rhim

Copyright copy 2014 Susana Machado 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

Recent success in the treatment of congenital blindness demonstrates the potential of ocular gene therapy as a therapeutic approachThe eye is a good target due to its small size minimal diffusion of therapeutic agent to the systemic circulation and low immuneand inflammatory responses Currently most approaches are based on viral vectors but efforts continue towards the synthesis andevaluation of new nonviral carriers to improve nucleic acid delivery Our objective is to evaluate the efficiency of novel cationicretinoic and carotenoic glycol phospholipids designated C20-18 C20-20 and C30-20 to deliver DNA to human retinal pigmentedepithelium (RPE) cells Liposomes were produced by solvent evaporation of ethanolic mixtures of the polyene compoundsand coformulated with 12-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) Addition of DNA to theliposomes formed lipoplexes which were characterized for binding size biocompatibility and transgene efficiency Lipoplexformulations of suitable size and biocompatibility were assayed for DNA delivery both qualitatively and quantitatively using RPEcells and a GFP-encoding plasmid The retinoic lipoplex formulation with DOPE revealed a transfection efficiency comparable tothe known lipid references 3120573-[N-(N1015840N1015840-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 12-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) and GeneJuice The results demonstrate that cationic polyene phospholipids have potential as DNAcarriers for ocular gene therapy

1 Introduction

Gene therapy is a promising treatment of several pathologies[1] and has been successfully employed to cure eye diseasesThe eye possesses several advantages for gene therapy (1)it is a small organ requiring only minor amounts of genecarriers (2) it is a spatially confined and compartmentalizedorgan (3) its tissues are readily accessible by microsurgical

techniques (4) it has transparent structures visualized bydirect noninvasive techniques (5) it is separated from sys-temic circulation by the blood-retinal barrier and therefore isan immune privileged organ (6) it offers a proximate control(the untreated eye) [2ndash4] (7) several eye pathologies aremonogenic and (8) mutations are identified

Successful gene therapy requires nucleic acid carriers ofminimal toxicity allowing long and stable gene expression

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 703253 13 pageshttpdxdoiorg1011552014703253

2 BioMed Research International

such as adenovirus and lentivirus [5] Nevertheless the useof viral gene carriers is limited by costly and time-consumingproduction restricted cloning capacity and most importantsevere immune responses [3 6] Nonviral carriers have thepotential to overcome these drawbacks although the effi-ciency of nonviral carriers still falls behind viral systems [2 47 8]Therefore there is a need to develop novel synthetic car-riers which package and protect the nucleic acid cargo suc-cessfully travel across the cytoplasmic membrane and ulti-mately release their nucleic acid load for nuclear uptake [5]

Nonviral carriers are typically cationic polymers or lipo-somes Cationic liposomes can easily be produced in largescale [9] can be made tissue specific have the ability to inter-act with biological membranes are well internalized by cellsdisplay low toxicity and are able to escape the endosome [7]

Cationic lipids interact with negatively charged nucleicacids through a combination of electrostatic and hydrophobicinteractions resulting in lipoplexes of multilamellar struc-tures [7] The structure and shape of the lipoplex and itslipidnucleic acid molar ratio are decisive for successful genedelivery [10]

The transfection efficiency and biocompatibility ofcationic lipid gene carriers can be altered by modificationsof the constituent parts of the lipids specifically the lipidbackbone (often glycerol) the hydrophilic head group andthe interconnecting linker Less diversity is reported for thehydrophobic part which is known to play a significant rolein the morphology of the lipoplex [4] The cationic headgroup is largely responsible for toxicity and determines thestrength of the electrostatic attraction for the negativelycharged nucleic acid Linker groups such as amides estersand ethers define the site for lipid cleavage which is againassociated with cytotoxicity [11] The hydrophobic domainusually consists of one or two saturated or monounsaturatedhydrocarbon chains

Dietary carotenoids are known to enhance visual perfor-mance and to reduce the risk of AMD progression [12] Theglycol lipids investigated in this report consist of retinoic(C205) and carotenoic (C309) polyene chains combinedwith a C200 and C180 chains (Figure 1) [13]

Our objective was to evaluate the gene delivery effi-ciency and cytotoxicity of the cationic polyene glycol lipidsagainst the reference glycerol lipids 3120573-[N-(N1015840N1015840-dimeth-ylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 12-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC referred toelsewhere as ldquodiC14-EPCrdquo [14]) (Figure 2) in human retinalpigmented epithelium (RPE) cells

The polyene lipids were formulated with either choles-terol (Chol) or 12-dioleoyl-sn-glycero-3-phosphoethanol-amine (DOPE) (Figure 3) and the impact of these colipidson gene delivery and cellular toxicity was assessed

2 Materials and Methods

21 GeneralMaterials TheARPE-19 cell line a human retinalpigment epithelial cell line was kindly provided by Dr Fran-scisco Ambrosio (Center for Neuroscience and Cell BiologyUniversity of Coimbra Portugal) Dulbeccorsquos modified eaglemedium (DMEM) culture medium and Dulbeccorsquos modified

eagle medium F12 ham (DMEM F12 Ham) culture mediumfetal bovine serum (FBS) trypsin glutamine penicillin-streptomycin solution dichloromethane thiazolyl blue tetra-zolium bromide (MTT) 004 N HCl in 2-propanol con-stituents dd-water tris-acetate-EDTA (TAE) constituentsAvertin anesthesia constituents (tribromoethanol) Triton X-100 Mowiol mounting media constituents sucrose (saccha-rose) paraformaldehyde eosin acetic acid dibutyl phthalatexylene (DPX) sodium phosphate potassium phosphate andgoat serum stock were purchased from Sigma-Aldrich (Por-tugal) Absolute ethanol was purchased fromMerckMillipore(Portugal)

PlasmidDNA containing the reporter gene green fluores-cent protein (GFP) was kindly provided by Dr Jean Bennett(University of Pennsylvania USA) Agarose was purchasedfrom Invitrogen (Portugal) GreenSafe and GeneJuice werepurchased from NZYTech (Portugal) For the phosphatebuffered saline (PBS) sodium chloride potassium chloride410158406-diamidino-2-phenylindole (DAPI) and optimal cuttingtemperature (OCT) cryostat embedding solution were pur-chased from VWR (Portugal) Primary antibody Iba1 amicroglia marker was purchased fromWako Pure ChemicalIndustries (Japan) and secondary antibody Alexa 594 waspurchased from Invitrogen (Portugal) Oxalic acid potas-sium permanganate (KMnO

4) and hematoxylin were pur-

chased fromMerck (Portugal) Control cationic lipids 3120573-[N-(N1015840N1015840-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 12-dimyristoyl-sn-glycero-3-ethylphosphocholine(EPC) [14] and the neutral colipids cholesterol (Chol) and 12-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) wereobtained from Avanti Polar Lipids (Alabaster AL USA)

22 Cationic Polyene Lipids C20-18 C20-20 and C30-20Three cationic polyene lipids C20-18 C20-20 and C30-20were synthesized from glycol retinoic acid (C205) or C30-acid (C309) and choline precursors as reported elsewhere[13 15]

23 Preparation of Lipid Ethanolic Stock Solutions Ethanolicstock solutions were made separately by first dissolvingeach cationic lipid and colipid in dichloromethane Thedichloromethane was removed under reduced pressure witha rotary evaporator and then the residue dissolved in absoluteethanol to a final lipid concentration of 1mM and subse-quently stored at minus80∘C

24 Preparation of Liposome Formulations Cholesterol andDOPE were employed as neutral colipids [4] Liposomesof C20-18Chol C20-20Chol C30-20Chol C20-18DOPEC20-20DOPE and C30-20DOPE in a lipidcolipid molarratio of 3 2 were generated by mixing ethanolic solutions ofcationic lipid and colipid and removing the ethanol underreduced pressureThe resulting thin filmswere then dissolvedin dd-water to a final lipid concentration of 2mM andincubated overnight at 4∘C Prior to use these hydratedliposome stocks were warmed to room temperature and thensubjected to ultrasounds for 30 minutes at 60∘C The knowncationic lipids DC-Chol and EPC were used as controls

BioMed Research International 3

C20-18

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

C20-20

C30-20

P

P

O

OP

Brminus

Brminus

Brminus

N+

N+

N+

Figure 1 C20-18 C20-20 and C30-20 cationic amphiphilic glycol polyene phospholipids The color of the polyene chain indicatesapproximately the visually appearance of the compounds Glycol backbone green hydrophilic part blue

25 Preparation of Lipoplex Formulations The addition ofpredetermined volumes of negatively charged plasmid DNAto positively charged liposomes resulted in lipoplex for-mulations of defined nitrogen-to-phosphorus (NP) molarcharge ratios DNA contained the reporter gene GFP Lipidconcentrations derived from a 2mM hydrated stock were0243mM 0162mM and 0081mM which correlates withthe molar charge ratios of 15 1 1 1 and 05 1 respectivelyEqual volumes of DNA solution in DMEM or DMEMF12Ham culture medium and lipids were mixed and incubatedat room temperature for 30min to allow lipoplex formation

26 Evaluation of Lipid-DNABinding Agel retardation assaywas used to evaluate lipoplex formation as a neutral ornet positive charge associated with the lipid-DNA complexretards migration through agarose gel Lipoplexes with vary-ing lipid DNA ratios were prepared as described above in atotal volume of 20120583L Samples were loaded onto a 1 agarosegel impregnated with GreenSafe and electrophoresed at 70Vin tris-acetate-EDTA (TAE) buffer The DNA bands wereobserved using an ultraviolet transilluminator

27 Physicochemical Characterization of the Lipoplexes Lip-oplexes were prepared in phosphate buffered solution (PBS)for subsequent determination of size and zeta potential bydynamic light scattering (DLS) and laser Doppler anemom-etry respectively using a Zetasizer Nanoseries ZS (MalvernInstruments) by diluting the sample 50x with dd-water

The size and size distribution of lipoplexes are indicatedas displayed by the instrument Measurement parameterswere as follows a laser wavelength of 633 nm and ascattering angle of 173∘ (fixed) The samples were loadedinto polystyrene cuvettes and three measurements wereperformed for which the mean result was recorded Particleswith PdI gt 07 and 119889H gt 2000 nm may be outside of theinstrumentrsquos reliable range

28 Evaluation of the In Vitro Cytotoxicity of LipoplexesD407 cells were maintained in DMEM culture mediumsupplemented with 5 FBS 1 glutamine and 1 penicillin-streptomycin solution at 37∘C with 5 CO

2 ARPE-19 cells

were maintained in DMEM F12 HAM culture mediumsupplemented with 10 FBS 1 glutamine and 1


Clminus

Clminus

+

N+

DC-Chol

EPC

O

O

O

N

O

O

OO

O

O

P

O

NH

Figure 2 Reference compounds DC-Chol and cationic glycerophosphospholipid EPC with two C140 chains Glycerol backbone greenhydrophilic parts blue

Clminus N+

O

O

O

O

O

OH

Chol

DOPEP

O

O

Ominus

Figure 3 Colipids Neutral cholesterol (Chol) and zwitterionic glycerophosphospholipid DOPE with two C181 chains Glycerol backbonegreen hydrophilic parts blue

penicillinstreptomycin at 37∘C with 5 CO2 For the

cytotoxicity assays cells were seeded at 35000 cells per well(D407) or 65000 cells per well (ARPE-19) in 24-well platesin complete medium and incubated for 24 h at 37∘C and5 CO

2 Cell seeding numbers differ with cell line due to

differences in cell doubling rate After 24 h cells were washedwith PBS and 250 120583L lipoplex formulations with various

NP molar charge ratios added to each well After a 4-hourincubation period 366120583L of culture medium was addedCells were incubated for 24 48 and 72 h Cell viability wasdetermined by the standard MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide) assay Briefly after theincubation period 50120583L of a 5mgmL MTT in PBS solutionwas added per well followed by a 4 h incubation period


after which the culture medium was removed and 500120583Lof a 004N HCl in 2-propanol solution was added per wellAfter dissolution of the formazan crystals their absorbancewas measured at 570 and 630 nm for each well Cell viabilitywas obtained by measurement of optical densities andnormalization against the positive control consisting of cellsgrown in standard culture medium

29 Evaluation of the In Vitro Transfection Efficiency ofLipoplexes Transfection was determined using cells seededat 100000 cells per well (D407) or 250000 cells per well(ARPE-19) in 6-well plates in complete growth mediumand incubated at 37∘C with 5 CO

2for 24 h Cell seeding

numbers differ with cell line due to differences in cell dou-bling rate Immediately prior to transfection cell monolayerswere washed with PBS The lipoplex formulations at variousNP molar charge ratios were added to each well in culturemedium up to 2mL and incubated for 4 h at 37∘C 5 CO

2

After this period the medium containing the lipoplexes wasremoved the cells were washed with PBS and completegrowth medium was added and further incubated for 72 h at37∘C 5 CO

2 Subsequently the cells were suspended using

trypsin washed with PBS and resuspended in 500120583L of PBSfor flow cytometry analysis (BD FACSCalibur Flow Cytome-ter BD Biosciences) Transfection efficiency was assessed bydetection of GFP with results normalized for 10000 events

210 Evaluation of the In Vivo Cytotoxicity of Lipoplexes Thein vivo compatibility of the C20-20DOPEDNA (NP molarcharge ratio 05 1) was evaluated by intravitreal injection ofthe lipoplexes dispersed in PBS into C57Bl6 mice C57BL6mice (6 months old) were housed at controlled temperaturewith 12 h lightdark cycle and food and water ad libitumAll experimental procedures involving animals were carriedout in accordance with the Portuguese and European Unionregulations (FELASA) and the Association for Researchin Vision and Ophthalmology (ARVO) All experimentalinjections were performed under anesthesia administeredby intraperitoneal injection (250mgkg dose) Intravitrealinjection was performed under a stereomicroscope (NikonStereoscopic Microscope) with a 30-gauge needle Only oneeye was injected the contralateral eye was left noninjected ascontrol Fourteen days postinjection themice were sacrificedby cervical dislocation and the eyes were enucleated andpreserved in PFA 4 at 4∘C until further use The eyeswere treated with 30 sucrose solution and embedded inOCT (Optimal Cutting Temperature) embedding solutionand stored at minus80∘C Cryosections of 10 120583m thickness wereused for detection of Iba1 (ionized calcium-binding adaptermolecule 1 a marker of active microglia used to detect localinflammation) by immunohistochemistry Cryosectionswerepermeabilized with PBS01 Triton X-100 and washed twicewith PBS A 1 1000 dilution of the Iba1 antibody was usedand the samples incubated overnight at 4∘C in the darkSamples were washed three times with PBS01 Triton X-100 and a 1 2000 dilution of the secondary antibody Alexa594 was used for detection and incubated for 1 h at roomtemperature in the dark Samples were washed three times

with PBS01 Triton x-100 mounted and visualized byfluorescencemicroscopy (Zeiss Axio Imager Z2 FluorescenceMicroscope) Samples were counterstained with DAPI (410158406-diamidino-2-phenylindole) a marker for nuclear detectionCryosections were also used for hematoxylin and eosinstaining to analyze the retina for its morphology and majorsigns of inflammation Sections were washed with distilledwater stained with hematoxylin for 1min washed withdistilled water stained with 1 eosin for 5min washed with02 acetic acid and mounted with DPX (dibutyl phthalatexylene) Samples were visualized under bright field (ZeissAxio Imager Z2 Fluorescence Microscope)

211 Statistical Analysis All figures are representative of atleast 3 separate experiments For comparison of multiple setsof data one-way ANOVA including the Dunnet post-testwas performed Results are expressed as mean +minus SEMStatistical analysis was performed using GraphPad Prism 6Software with 119875 lt 005 considered statistically significant

3 Results

31 Polyene Lipids Form Stable Lipid-DNA Complexes(Lipoplexes) The size and surface charge of lipid-DNAcomplexes composed of the polyene lipids were determinedusing Dynamic Light Scattering (DLS) (Table 1) Thelipoplexes were prepared in PBS since this was the vehicleused for further experiments The average size range oflipoplexes composed of reference lipids DC-Chol and EPCwas 119889H (hydrodynamic diameter) = 320ndash2010 nm whilefor those composed of C20-18 C20-20 and C30-20 was119889H = 320ndash2210 nm The average size of the lipoplexes didnot correlate with increasing NP ratio with the exceptionof EPCDOPE and C20-18Chol (denoted by lowast) and alsodid not correlate with formulation of the same lipid withdifferent colipids Chol and DOPE with the exceptionof EPC and C30-20 lipoplexes (denoted by dagger) DLS is atechnique that depends on the scattering of light with theparticles and polydispersity may ldquohiderdquo real populations dueto the presence of large particles Table 1 shows the modewhich is the most frequent size in the samples If samplesare monodisperse 119889H values are representative of the sampleand will be similar to the mode However if the sample ispolydisperse 119889H values represent less accurately the sampleand its value is apart from the mode [16]

The surface charge values (as measured by zeta potentialZP Table 1) indicate stable particles The lipoplexes of thereference lipids revealedZP = minus54plusmn2ndashminus69plusmn3mVwhereas thepolyene lipoplexes revealed a broader range with ZP = minus30 plusmn2ndash minus66 plusmn 1mVThe ZP ranges did not correlate with particlesize as the reference lipids were found to have a narrow ZPrange yet generally broader particle distribution comparedwith the polyene lipoplexes Significant size changes were notassociated with significant changes in ZP Furthermore ZPvalues are not correlated with increasing NP ratio except forC30-20Chol

The cationic liposomes were combined with a GFP-codedplasmid DNA to evaluate relative gene transfer efficiency toRPE cells The DNA binding efficiency of C20-18 C20-20


Table 1 Size and surface charge (as measured by zeta potential ZP) of C20-18 C20-20 C30-20 DC-Chol and EPC lipoplexes in PBS atvarying NP molar charge ratios with either Chol or DOPE as colipids

Lipid Colipid 119873119875 ratio dH (nm) Mode (nm) Pdl ZP (mV)

DC-Chol

05 1 1180 477 0718 minus55 plusmn 561

Chol 1 1 1480 455 0840dagger minus55 plusmn 519

15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065

DOPE 1 1 1310 600 0424 minus69 plusmn 276

15 1 1290 506 0768lowast minus65 plusmn 106

EPC

05 1 510dagger 488 0220dagger minus54 plusmn 165

Chol 1 1 360 380 0058 minus57 plusmn 101

15 1 320dagger 322dagger 0234dagger minus57 plusmn 085

05 1 1410 528 0750 minus57 plusmn 122


15 1 2010lowast 656 0862 minus54 plusmn 198

C20-18

05 1 320 256 0322 minus41 plusmn 1081


15 1 1523lowast 490 0702lowast minus43 plusmn 811

05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20

05 1 500dagger 355lowastdagger 0353 minus36 plusmn 143lowast

Chol 1 1 820 763 0178 minus60 plusmn 186dagger

15 1 710 697 0260 minus66 plusmn 058dagger

05 1 1500 937lowast 0144 minus30 plusmn 171


15 1 1140 768 0220 minus40 plusmn 012daggerComparison between Chol and DOPE formulations of one lipid with the samemolar charge ratio lowastComparison between ratios (05 1 versus 1 1 or 1 1 versus15 1) of the same lipid formulation

C30-20 DC-Chol and EPC-containing lipoplexes at variousNP molar charge ratios was assessed using a gel retardationassayThenegatively charged plasmidDNAwhen interactingwith sufficient cationic lipids forms a neutral or net positivecomplex that will not migrate through the agarose gel Asseen in Figure 4 all formulations revealed partial or totalDNA retention demonstrating cationic lipidDNA lipoplexformation An increase in NP molar charge ratio resulted inan increase inDNA retention C20-18Chol and C30-20Cholformulations revealed a higher degree of complexation withDNA as revealed by a diminished DNA migration bandwhen compared with C20-18 C20-20 and C30-20 formula-tions (Figures 4(a) and 4(b)) The reference lipids DC-Choland EPC showed a similar trend with higher DNA retentionwith increasing NP molar ratios (Figures 4(c) and 4(d))Our plasmid binding results are in accordance with previousoutcomes [13 15] where C20-20 and C30-20 lipoplexes

formulated with DNA also revealed partial retention in a gelretardation assay at low NP charge ratios

32 Toxicity of the Lipoplexes Is Concentration Dependentin RPE Cells ARPE-19 cells were cultured in the presenceof C20-18 C20-20 C30-20 DC-Chol and EPC lipoplexesat varying NP molar charge ratios and cell viability wasassessed after 24 48 and 72 h (Figures 5 6 7 8 and 9)A decrease in cell viability below 75 was defined as thecytotoxicity threshold according to the ISO standard forbiological evaluation of medical devices [17]

Toxicity was concentration dependent (increasing withincreased NP molar charge ratio) and time dependent(duration of the incubation period) as previously described[18] Lipoplex C20-20 was found to be more cytotoxic thanC20-18 and C30-20 The reference lipids exhibit toxicitycomparable with C20-20


C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20

DOPE Chol DOPE CholL DNA 05 1 1 1 1 115 1 15 105 1 1 1 15 105 1 1 1 15 105 1

(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)

Figure 4 Gel retardation assays with lipoplexes C20-18 (a) C20-20 and C30-20 (b) DC-Chol (c) and EPC (d) with DOPE and Chol ascolipids Molar charge ratios NP used were 05 1 1 1 and 15 1 Retention of DNA increases with increasing molar charge ratios

Based on the cytotoxicity results the 05 1 NP molarcharge ratio was subsequently used in transfection assays andin vivo assays for all formulations due to lower cytotoxicity

33 Toxicity of C20-20DOPE Lipoplex under In Vivo Con-ditions To evaluate whether the level of toxicity observedin vitro was similar for in vivo C20-20DOPE lipoplexesprepared in PBS at a NP molar charge ratio of 05 1 wereadministered by intravitreal injection to the eyes of C57BL6mice After 14 days mice were sacrificed and the eyes wereenucleated and used for Iba1 detection by immunohisto-chemistry or stained with hematoxylin and eosin for assess-ment of retinal integrity and inflammation Iba1 is a calcium-binding protein characteristic of microglia that is expressedwhen the microglia is reactive that is in situations whereinflammation occurs [19] Our results showed that retinasof mice injected with C20-20DOPE lipoplexes at the NPmolar charge ratio 05 1 (Figure 10) revealed microglia in thenonreactive state with short cellular ramifications indicating

the absence of inflammation in the retinaThe control whichis the contralateral noninjected eye also showed similarmicroglia morphology in its nonreactive state These resultsconfirm that C20-20 is not toxic when administered in vivoin the retina of C57Bl6 mice

Administration of drugs by intravitreal injection canresult in morphological disruption of the retinal structurecaused by the injection procedure and tissue inflammationHematoxylin and eosin staining (Figure 11) showed no mor-phological disruption of the retina in the in vivo study

34 Transfection Efficiency of C20-18 C20-20 and C30-20 Lipoplexes in RPE Cells We investigated the in vitrotransfection efficiency of the C20-18 C20-20 and C30-20lipoplexes in retinal cells using a plasmid coding for GFPand assessing its expression qualitatively by fluorescencemicroscopy and quantitatively by flow cytometry ARPE-19 cells were transfected with C20-18 C20-20 and C30-20lipoplexes with either Chol orDOPE as colipid at aNPmolar


0

20

40

60

100

120

8075

Viab

ility

()

lowastlowastlowastlowast lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast


lowastlowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h

Figure 5 DC-Chol lipoplex cytotoxicity ARPE-19 cell viability forDC-Chol lipoplexes with either Chol or DOPE as colipid at variousNP ratios incubated up to 72 h Horizontal line at 75 viabilityrepresent the threshold according to the ISO standard for in vitrotesting of biological devices C+ represents untreated cells and Cminusrepresents cells treated to induce cell death Statistical significance(lowast) of 95 (119875 lt 005)

0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h

Figure 6 EPC lipoplex cytotoxicity ARPE-19 cell viability for EPClipoplexes with either Chol or DOPE as colipids at various NPmolar charge ratios incubated up to 72 h Horizontal line at 75viability represents the threshold according to the ISO standardfor in vitro testing of biological devices C+ represents untreatedcells and Cminus represents cells treated to induce cell death Statisticalsignificance (lowast) of 95 (119875 lt 005)

ratio of 05 1 transfection was evaluated by GFP expressionafter 72 h (Figures 12 and 13 and Supplementary Figure S2available online at httpdxdoiorg1011552014703253) Forpolyene and reference lipids alike all DOPE formulatedlipoplexes with the exception of C30-20 revealed greatertransfection efficiency than those formulations with Cholas colipid (Figure 13) Of the polyene lipids studied the

0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast

lowastlowastlowastlowastlowast

24h48h

72h

Figure 7 C20-18 lipoplex cytotoxicity ARPE-19 cell viability forC20-18 lipoplexes with either Chol or DOPE as colipid at variousNP ratios incubated up to 72 h Horizontal line at 75 viabilityrepresents the threshold according to the ISO standard for in vitrotesting of biological devices C+ represents untreated cells and Cminusrepresents cells treated to induce cell death Statistical significance(lowast) of 95 (119875 lt 005)

0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol DOPE

C20-20








lowastlowastlowast lowastlowastlowastlowastlowastlowast

24h48h

72h


C20-20DOPE formulation revealed the greatest transfectionefficiency with an average of 8 followed by C20-18DOPEwith 6 (Figure 13) Upon comparison of the polyenelipoplexes with DC-Chol and EPC lipoplexes C20-18 andC20-20 lipoplexes revealed greater competitive transfectionefficiencies whenChol was used as colipid (Figures 12 and 13)



C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h


The transfection efficiency of C20-20 and C30-20lipoplexes at NP molar charge ratio of 05 1 containingeither Chol or DOPE as colipid was also assessed in D407cells against the positive control GeneJuice (supplementaryFigure S1) As was found with the ARPE-19 cell line the C20-20DOPE formulation outperformed the correspondingChol formulation in the D407 cell line (supplementaryFigure S1) In addition C20-20DOPE revealed a level oftransfection competitive to the control GeneJuice whereasthe C30-20 lipid revealed only a low level of transfectionwith Chol as colipid

4 Discussion

Cationic polyene lipids were evaluated for their potential tocarry DNA into retinal cells Transfection efficiency was cor-related with molecular structure lipoplex size zeta potentialbiocompatibility and the presence of colipids DOPE andcholesterol increase DNA delivery and thus were employedas colipids DOPE often referred to as a ldquofusogenic lipidrdquocould alter the packing of the hydrophobic chains withinthe lipoplex [10] by promoting a nonlamellar structure thusdestabilizing the endosomal membrane and releasing DNA[7] upon membrane fusion between the lipoplex and theendosome [8] The addition of plasmid DNA to the self-aggregated liposomes formed lipoplexes which were char-acterized for their physical properties and capacity of DNAcomplexation All liposomes showed some degree of com-plexation with plasmid DNA however complete retentionof the lipoplexes in the gel retardation assay was not alwaysobserved With the exception of C20-20 the polyene lipidscomplexed DNA more effectively when cholesterol was used

as colipid while EPC and DC-Chol showed more completebinding with formulations that contained DOPE (Figure 2)It was expected that increasing the NP molar ratio for thoselipoplex formulations with incomplete gel retardation wouldresult in complete DNA retention [20] at the expense of cellviability

The analysis of the physical properties of the lipoplexessummarized in Table 1 shows that some EPC and DC-Cholformulations gave rise to large lipoplex sizes and high poly-dispersity index (PdI) This is most likely due to aggregationand fusion of liposomes during lipoplex formation [21]Despite this observation the sizes of the lipoplexes are in therange of those generally considered adequate for in vitro cellinternalization although a correlation between size surfacecharge and transfection efficiency is still debatable [10]

Almofti et al demonstrated for the cationic lipid DC-6-14that lipoplex size increased lipoplex transfection [22] Withthe exception of DC-Chol all DOPE formulations revealedbetter transfection efficiencies with larger lipoplex comparedto Chol formulations This can be explained by the multipleendocytic mechanisms existing in the cells for internalizationof lipoplexes of different sizes [7]

Zeta potential reflects the surface charge and lipoplexstability ZP gt plusmn30mV indicates stable particles andZP lt plusmn10mV suggests unstable particles with a tendency toaggregation [10]

Our lipoplexes presented negative zeta potential valuesbetween minus30 plusmn 2 and minus66 plusmn 1mV Although a positivelycharged lipoplex interacts more favorably with the negativecharged biological membranes it is not a necessary conditionfor transfection [9] and as shown in Figures 12 and 13 C20-20DOPE andC20-18DOPE lipoplexes were able to transfectretinal cells

Negative ZP values most likely derive from DNA boundto the outer shell of the lipoplexes and from the phosphategroups associated with the solvent (PBS) that remains closeto the surface of the lipoplexes

Successful gene carriers do not only transfect efficientlybut they also need to be biocompatible with the organismand target tissue In vitro testing showed that the lipoplexeshave increased in vitro toxicity with increasing NP molarratio and incubation period in line with reference lipidsDC-Chol EPC and other cationic lipids [18 23] Toxicityis lipoplex specific and tissue specific dose dependent androute dependent Lipoplex formulations can be modified toreduce toxicity or to promote transfection efficiency oneexample is PEGylation which can control lipoplex size andincrease circulation time in vivo [7] In the retina toxicity andinflammation can be evaluated by morphological analysisof the retina and by immunohistochemistry detection ofinflammatory markers such as the microglia The microgliaconsists of macrophages and monocytes and is a normalconstituent in the retina mainly distributed in the nerve fiberlayer and ganglion cell layer [24] In its nonreactive stateramified microglia can be observed in the tissue Howeverwhen tissue is damaged microglial cells migrate to all retinallayers retract cell processes (ramifications) and increasecell size [25] Our results show in vivo compatibility ofC20-20 with the retinal tissue as shown by the absence of


InjectedC20-20dopeDNA

Injected and electroporatedC20-20dopeDNA

Noninjected50120583m

Figure 10 Immunohistochemistry detection of Iba1 in mouse retinas injected with C20-20DOPE lipoplexes at the NP molar charge ratio05 1 Magnification 100x and scale bar 50 120583m




Noninjected and electroporated

Figure 11 Hematoxylin and eosin staining of mouse retinas injected with C20-20DOPE lipoplexes at the NP molar charge ratio 05 1showing the maintenance of the integrity of the retinal layered structure Magnification 100x and scale bar 50 120583m

morphological alterations in the retina and by nonreactivemicroglia

The efficiency of in vitro gene delivery was evaluatedwith a plasmid coding for the GFP reporter gene throughdetection of GFP-positive cells C20-20 and the C20-18formulations showed greater transfection efficiency relativeto C30-20 when either DOPE or Chol was used as colipid Alllipid-DOPE formulations showed enhanced transfection effi-ciency when compared with lipid-Chol formulations DOPEprobably facilitates lipoplex phase transitions from lamellarto inverted hexagonal packing which allows an effectivefusion of the lipoplex and the cellular endocytic membrane[26 27] It has previously been demonstrated that lipoplexesof EPC C20-20 and C30-20 with either DOPE or Chol gaverise to lamellar morphologies as shown by small-angle X-ray scattering [12] However only those lamellar lipoplexescontainingDOPE are expected to invert to hexagonal packingin the endosome

Although transfection in ARPE-19 cells was higher forthe DC-CholDOPE and EPCDOPE lipoplexes transfec-tion efficiencies of the C20-18DOPE and C20-20DOPElipoplexes are noteworthy when compared to other investi-gations reporting considerably lower efficiencies [28]

In an attempt to further compare the relative transfectionefficiency of C20-20 and C30-20 we tested the same for-mulations in D407 cells (supplementary Figure S1) anothercell line from the retina but with a faster mitotic rate thanARPE-19 We observed with these cells a greater transfectionefficiency than with ARPE-19 cells which is most likely dueto the higher mitotic rate since during mitosis the nuclear

membrane is disaggregated and therefore nuclear penetrationof nucleic acids is facilitated

Remarkably C20-20 displayed greater transgene effi-ciency than C30-20 in both cell lines tested The retinoiclipids C20-18 and C20-20 may therefor enter the nucleus viaa possible retinoid receptor [29] Further studies are requiredto investigate a possible retinoid receptor mediated nuclearuptake mechanism

C20-18Chol and C20-20Chol performed comparablyto references DC-Chol and EPC C20-20DOPE lipoplexesdelivered DNA in vitro to RPE cells and were biocompatibleduring in vivo applications These results encourage us tooptimize and characterize this process

5 Conclusion

Cationic liposomes formulated from polyene lipids C20-18C20-20 and C30-20 and a neutral colipid were combinedwith a GFP-coded plasmid DNA for studies in retina cellsGel electrophoresis was used to confirm the formation oflipoplexes in all formulations containing either DOPE orChol as colipid The diameters of lipoplexes were relativelylarge and variable however this did not appear to hindertransfection since for a given cationic lipid larger lipoplexesgave rise to greater transfection (Table 1 and Figure 13)Toxicity associated with the polyene lipids under in vitroconditions was as anticipated concentration dependent andincreased with increasing NP ratio Toxicity was also foundto increase with increasing incubation period in line with theformulations for reference lipids DC-Chol and EPC


Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20

Figure 12 Qualitative assessment of lipoplex transfection ARPE-19 cells were incubated with lipoplexes containing lipids C20-18 C20-20 orC30-20 against reference lipids DC-Chol and EPC with either Chol or DOPE as colipid at NP molar ratio 05 1 for 4 h and GFP-expressingcells evaluated by fluorescence microscopy after 72 h Magnification 100x and scale bar 100 120583m

12 BioMed Research InternationalG

FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast

Figure 13 The transfection efficiency of lipoplexes ARPE-19 cellswere incubated with lipoplexes containing lipids C20-18 C20-20or C30-20 against reference lipids DC-Chol and EPC with eitherChol or DOPE as colipid at NP molar ratio 05 1 for 4 h andtransfection efficiencies measured by GFP expression determinedby flow cytometry after 72 h Statistical significance (lowast) of 95 (119875 lt005)

C20-20 was found to have greater in vitro cytotoxicitythan C20-18 or C30-20 Nonetheless C20-20 showed noevidence of inflammation in the mouse retina In vitro trans-fection efficiencies of 6 and 8 were achieved with ARPE-19 cells with C20-18DOPE and C20-20DOPE respectivelyFurthermore DNA delivery using C20-20DOPE outper-formed C30-20 in D407 retinal cells and revealed a level oftransfection competitive to reference GeneJuice

Overall C20-20DOPE gave promising results for oculargene therapy Future work is directed to optimize formula-tions for in vivo ocular gene delivery

Conflict of Interests

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

Acknowledgments

TheARPE-19 cell line a human retinal pigment epithelial cellline was kindly provided byDr FransciscoAmbrosio (Centerfor Neuroscience and Cell Biology University of CoimbraPortugal) Plasmid DNA containing the reporter gene greenfluorescent protein (GFP) andD407 cell line a human retinalpigment epithelial cell line was kindly provided by Dr JeanBennett (University of Pennsylvania USA) This publicationwasmade possible by grants fromTheFP7 programunder theMarie Curie Scheme PIRG-GA-2009-249314 FCT Portugal(IBBLA PEst-OEEQBLA00232011 SFRHBD768732011to Sofia Calado SFRHBD703182010 to Ana VanessaOliveira and PTDCSAU-BEB09847542008 to Gabriela ASilva) and from the Qatar National Research Fund under

the National Priorities Research Program award NPRP08-705-3-144 (LPI M Pungente) Its contents are solely theresponsibility of the authors and do not necessarily representthe official views of the Qatar National Research Fund

References

[1] T A Balbino A AM Gasperini C L P Oliveira A R AzzoniL P Cavalcanti and L G de la Torre ldquoCorrelation of thephysicochemical and structural properties of pDNAcationicliposome complexes with their in vitro transfectionrdquo Langmuirvol 28 no 31 pp 11535ndash11545 2012

[2] J W B Bainbridge A R Mistry A J Thrasher and R R AlildquoGene therapy for ocular angiogenesisrdquo Clinical Science vol104 no 6 pp 561ndash575 2003

[3] S M Conley X Cai and M I Naash ldquoNonviral ocular genetherapy assessment and future directionsrdquo Current Opinion inMolecular Therapeutics vol 10 no 5 pp 456ndash463 2008

[4] S M Conley and M I Naash ldquoNanoparticles for retinal genetherapyrdquo Progress in Retinal and Eye Research vol 29 no 5 pp376ndash397 2010

[5] Y Shan T Luo C Peng et al ldquoGene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectorsrdquo Biomaterialsvol 33 no 10 pp 3025ndash3035 2012

[6] V Tamboli G P Mishra and A K Mitra ldquoPolymeric vectorsfor ocular gene deliveryrdquoTherapeutic Delivery vol 2 no 4 pp523ndash536 2011

[7] Z U Rehman I S Zuhorn and D Hoekstra ldquoHow cationiclipids transfer nucleic acids into cells and across cellular mem-branes Recent advancesrdquo Journal of Controlled Release vol 166no 1 pp 46ndash56 2013

[8] N Nayerossadat T Maedeh and P A Ali ldquoViral and non-viral delivery systems for gene deliveryrdquo Advanced BiomedicalResearch vol 1 article 27 2012

[9] M Ramezani M Khoshhamdam A Dehshahri and BMalaekeh-Nikouei ldquoThe influence of size lipid compositionand bilayer fluidity of cationic liposomes on the transfectionefficiency of nanolipoplexesrdquo Colloids and Surfaces B Biointer-faces vol 72 no 1 pp 1ndash5 2009

[10] B Ma S Zhang H Jiang B Zhao and H Lv ldquoLipoplexmorphologies and their influences on transfection efficiency ingene deliveryrdquo Journal of Controlled Release vol 123 no 3 pp184ndash194 2007

[11] D Pezzoli R Chiesa L de Nardo and G Candiani ldquoWe stillhave a long way to go to effectively deliver genesrdquo Journal ofApplied Biomaterials and Fundamental Materials vol 10 no 2pp e82ndashe91 2012

[12] E Loskutova J Nolan A Howard and S Beatty ldquoMacularpigment and its contribution to visionrdquo Nutrients vol 5 no 6pp 1962ndash1969 2013

[13] L J Popplewell A Abu-Dayya T Khanna et al ldquoNovel cationiccarotenoid lipids as delivery vectors of antisense oligonu-cleotides for exon skipping in duchenne muscular dystrophyrdquoMolecules vol 17 no 2 pp 1138ndash1148 2012

[14] R Koynova B Tenchov L Wang and R C MacDonaldldquoHydrophobicmoiety of cationic lipids stronglymodulates theirtransfection activityrdquoMolecular Pharmaceutics vol 6 no 3 pp951ndash958 2009

[15] C L Z M Oslashpstad A Zaidi H R Sliwka et al ldquoNovel cationicpolyene glycol phospholipids as DNA transfer reagents lack


of a structure-activity relationship due to uncontrolled self-assembling processesrdquo Chemistry and Physics of Lipids vol 183pp 117ndash136 2014

[16] E Tomaszewska K Soliwoda K Kadziola et al ldquoDetectionlimits of DLS and UV-Vis spectroscopy in characterization ofpolydisperse nanoparticles colloidsrdquo Journal of Nanomaterialsvol 2013 Article ID 313081 10 pages 2013

[17] ISO 10993-52009 Biological evaluation of medical devicesmdashpart 5 tests for in vitro cytotoxicity

[18] H Lv S Zhang B Wang S Cui and J Yan ldquoToxicity ofcationic lipids and cationic polymers in gene deliveryrdquo Journalof Controlled Release vol 114 no 1 pp 100ndash109 2006

[19] A M Santos R Calvente M Tassi et al ldquoEmbryonic andpostnatal development of microglial cells in the mouse retinardquoJournal of Comparative Neurology vol 506 no 2 pp 224ndash2392008

[20] J Yeh N A Khalique L Raju et al ldquoSynthesis self-assemblyand lipoplex formulation of two novel cyclic phosphonatelipidsrdquo QScience Connect vol 2012 article 6 2012

[21] C L Oslashpstad H Sliwka V Partali et al ldquoSynthesis self-assembling and gene delivery potential of a novel highlyunsaturated conjugated cationic phospholipidrdquo Chemistry andPhysics of Lipids vol 170-171 pp 65ndash73 2013

[22] M R Almofti H Harashima Y Shinohara A Almofti W Liand H Kiwada ldquoLipoplex size determines lipofection efficiencywith or without serumrdquo Molecular Membrane Biology vol 20no 1 pp 35ndash43 2003

[23] C R Dass ldquoCytotoxicity issues pertinent to lipoplex-mediatedgene therapy in-vivordquo Journal of Pharmacy and Pharmacologyvol 54 no 5 pp 593ndash601 2002

[24] X Zeng Y Ng and E Ling ldquoNeuronal and microglial responsein the retina of streptozotocin-induced diabetic ratsrdquo VisualNeuroscience vol 17 no 3 pp 463ndash471 2000

[25] E Ulbricht T Pannicke S Uhlmann P Wiedemann AReichenbach and M Francke ldquoActivation of retinal microglialcells is not associated with Muller cell reactivity in vitrec-tomized rabbit eyesrdquo Acta Ophthalmologica vol 91 no 1 ppe48ndashe55 2013

[26] H Farhood N Serbina and L Huang ldquoThe role of dioleoylphosphatidylethanolamine in cationic liposome mediated genetransferrdquo Biochimica et Biophysica Acta vol 1235 no 2 pp 289ndash295 1995

[27] M Vidal and D Hoekstra ldquoIn vitro fusion of reticulocyte endo-cytic vesicles with liposomesrdquo Journal of Biological Chemistryvol 270 no 30 pp 17823ndash17829 1995

[28] A V Oliveira A P Silva D B Bitoque G A Silva andA M Rosa Da Costa ldquoTransfection efficiency of chitosanand thiolated chitosan in retinal pigment epithelium cells acomparative studyrdquo Journal of Pharmacy and Bioallied Sciencesvol 5 no 2 pp 111ndash118 2013

[29] N Bushue and Y-J Y Wan ldquoRetinoid pathway and cancertherapeuticsrdquo Advanced Drug Delivery Reviews vol 62 no 13pp 1285ndash1298 2010

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Polymer ScienceInternational Journal of


CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

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


Volume 2014

MaterialsJournal of

Nano

materials


Journal ofNanomaterials


such as adenovirus and lentivirus [5] Nevertheless the useof viral gene carriers is limited by costly and time-consumingproduction restricted cloning capacity and most importantsevere immune responses [3 6] Nonviral carriers have thepotential to overcome these drawbacks although the effi-ciency of nonviral carriers still falls behind viral systems [2 47 8]Therefore there is a need to develop novel synthetic car-riers which package and protect the nucleic acid cargo suc-cessfully travel across the cytoplasmic membrane and ulti-mately release their nucleic acid load for nuclear uptake [5]

Nonviral carriers are typically cationic polymers or lipo-somes Cationic liposomes can easily be produced in largescale [9] can be made tissue specific have the ability to inter-act with biological membranes are well internalized by cellsdisplay low toxicity and are able to escape the endosome [7]

Cationic lipids interact with negatively charged nucleicacids through a combination of electrostatic and hydrophobicinteractions resulting in lipoplexes of multilamellar struc-tures [7] The structure and shape of the lipoplex and itslipidnucleic acid molar ratio are decisive for successful genedelivery [10]

The transfection efficiency and biocompatibility ofcationic lipid gene carriers can be altered by modificationsof the constituent parts of the lipids specifically the lipidbackbone (often glycerol) the hydrophilic head group andthe interconnecting linker Less diversity is reported for thehydrophobic part which is known to play a significant rolein the morphology of the lipoplex [4] The cationic headgroup is largely responsible for toxicity and determines thestrength of the electrostatic attraction for the negativelycharged nucleic acid Linker groups such as amides estersand ethers define the site for lipid cleavage which is againassociated with cytotoxicity [11] The hydrophobic domainusually consists of one or two saturated or monounsaturatedhydrocarbon chains

Dietary carotenoids are known to enhance visual perfor-mance and to reduce the risk of AMD progression [12] Theglycol lipids investigated in this report consist of retinoic(C205) and carotenoic (C309) polyene chains combinedwith a C200 and C180 chains (Figure 1) [13]

Our objective was to evaluate the gene delivery effi-ciency and cytotoxicity of the cationic polyene glycol lipidsagainst the reference glycerol lipids 3120573-[N-(N1015840N1015840-dimeth-ylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 12-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC referred toelsewhere as ldquodiC14-EPCrdquo [14]) (Figure 2) in human retinalpigmented epithelium (RPE) cells

The polyene lipids were formulated with either choles-terol (Chol) or 12-dioleoyl-sn-glycero-3-phosphoethanol-amine (DOPE) (Figure 3) and the impact of these colipidson gene delivery and cellular toxicity was assessed

2 Materials and Methods

21 GeneralMaterials TheARPE-19 cell line a human retinalpigment epithelial cell line was kindly provided by Dr Fran-scisco Ambrosio (Center for Neuroscience and Cell BiologyUniversity of Coimbra Portugal) Dulbeccorsquos modified eaglemedium (DMEM) culture medium and Dulbeccorsquos modified

eagle medium F12 ham (DMEM F12 Ham) culture mediumfetal bovine serum (FBS) trypsin glutamine penicillin-streptomycin solution dichloromethane thiazolyl blue tetra-zolium bromide (MTT) 004 N HCl in 2-propanol con-stituents dd-water tris-acetate-EDTA (TAE) constituentsAvertin anesthesia constituents (tribromoethanol) Triton X-100 Mowiol mounting media constituents sucrose (saccha-rose) paraformaldehyde eosin acetic acid dibutyl phthalatexylene (DPX) sodium phosphate potassium phosphate andgoat serum stock were purchased from Sigma-Aldrich (Por-tugal) Absolute ethanol was purchased fromMerckMillipore(Portugal)

PlasmidDNA containing the reporter gene green fluores-cent protein (GFP) was kindly provided by Dr Jean Bennett(University of Pennsylvania USA) Agarose was purchasedfrom Invitrogen (Portugal) GreenSafe and GeneJuice werepurchased from NZYTech (Portugal) For the phosphatebuffered saline (PBS) sodium chloride potassium chloride410158406-diamidino-2-phenylindole (DAPI) and optimal cuttingtemperature (OCT) cryostat embedding solution were pur-chased from VWR (Portugal) Primary antibody Iba1 amicroglia marker was purchased fromWako Pure ChemicalIndustries (Japan) and secondary antibody Alexa 594 waspurchased from Invitrogen (Portugal) Oxalic acid potas-sium permanganate (KMnO

4) and hematoxylin were pur-

chased fromMerck (Portugal) Control cationic lipids 3120573-[N-(N1015840N1015840-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and 12-dimyristoyl-sn-glycero-3-ethylphosphocholine(EPC) [14] and the neutral colipids cholesterol (Chol) and 12-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) wereobtained from Avanti Polar Lipids (Alabaster AL USA)

22 Cationic Polyene Lipids C20-18 C20-20 and C30-20Three cationic polyene lipids C20-18 C20-20 and C30-20were synthesized from glycol retinoic acid (C205) or C30-acid (C309) and choline precursors as reported elsewhere[13 15]

23 Preparation of Lipid Ethanolic Stock Solutions Ethanolicstock solutions were made separately by first dissolvingeach cationic lipid and colipid in dichloromethane Thedichloromethane was removed under reduced pressure witha rotary evaporator and then the residue dissolved in absoluteethanol to a final lipid concentration of 1mM and subse-quently stored at minus80∘C

24 Preparation of Liposome Formulations Cholesterol andDOPE were employed as neutral colipids [4] Liposomesof C20-18Chol C20-20Chol C30-20Chol C20-18DOPEC20-20DOPE and C30-20DOPE in a lipidcolipid molarratio of 3 2 were generated by mixing ethanolic solutions ofcationic lipid and colipid and removing the ethanol underreduced pressureThe resulting thin filmswere then dissolvedin dd-water to a final lipid concentration of 2mM andincubated overnight at 4∘C Prior to use these hydratedliposome stocks were warmed to room temperature and thensubjected to ultrasounds for 30 minutes at 60∘C The knowncationic lipids DC-Chol and EPC were used as controls


C20-18

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

C20-20

C30-20

P

P

O

OP

Brminus

Brminus

Brminus

N+

N+

N+







2 ARPE-19 cells



Clminus

Clminus

+

N+

DC-Chol

EPC

O

O

O

N

O

O

OO

O

O

P

O

NH


Clminus N+

O

O

O

O

O

OH

Chol

DOPEP

O

O

Ominus












2







3 Results







DC-Chol

05 1 1180 477 0718 minus55 plusmn 561


15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065



EPC




05 1 1410 528 0750 minus57 plusmn 122



C20-18

05 1 320 256 0322 minus41 plusmn 1081



05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20












C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




C20-18

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

C20-20

C30-20

P

P

O

OP

Brminus

Brminus

Brminus

N+

N+

N+







2 ARPE-19 cells



Clminus

Clminus

+

N+

DC-Chol

EPC

O

O

O

N

O

O

OO

O

O

P

O

NH


Clminus N+

O

O

O

O

O

OH

Chol

DOPEP

O

O

Ominus












2







3 Results







DC-Chol

05 1 1180 477 0718 minus55 plusmn 561


15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065



EPC




05 1 1410 528 0750 minus57 plusmn 122



C20-18

05 1 320 256 0322 minus41 plusmn 1081



05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20












C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




Clminus

Clminus

+

N+

DC-Chol

EPC

O

O

O

N

O

O

OO

O

O

P

O

NH


Clminus N+

O

O

O

O

O

OH

Chol

DOPEP

O

O

Ominus












2







3 Results







DC-Chol

05 1 1180 477 0718 minus55 plusmn 561


15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065



EPC




05 1 1410 528 0750 minus57 plusmn 122



C20-18

05 1 320 256 0322 minus41 plusmn 1081



05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20












C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials








2







3 Results







DC-Chol

05 1 1180 477 0718 minus55 plusmn 561


15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065



EPC




05 1 1410 528 0750 minus57 plusmn 122



C20-18

05 1 320 256 0322 minus41 plusmn 1081



05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20












C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials






DC-Chol

05 1 1180 477 0718 minus55 plusmn 561


15 1 1220 516 0611 minus65 plusmn 311

05 1 900 441 0664 minus63 plusmn 065



EPC




05 1 1410 528 0750 minus57 plusmn 122



C20-18

05 1 320 256 0322 minus41 plusmn 1081



05 1 550 487 0425 minus53 plusmn 673


15 1 1270 453 0596 minus36 plusmn 174

C20-20

05 1 560 548 0130 minus53 plusmn 027


15 1 660 537 0257 minus61 plusmn 037

05 1 640 465 0304 minus53 plusmn 143


15 1 750 594 0147 minus62 plusmn 050

C30-20












C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




C20-18

DOPE

L DNA 05 1 1 1 15 1

Chol

L DNA 05 1 1 1 15 1

(a)

C20-20 C30-20


(b)

DC-CholDOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(c)

EPC

DOPE Chol

L DNA 05 1 1 1 1 115 1 15 105 1

(d)








0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




0

20

40

60

100

120

8075

Viab

ility

()





C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

DC-Chol

DOPE

24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast

C+ Cminus DNA 05 105 1 1 11 1 15 115 1Chol

EPCDOPE

lowastlowastlowast

lowastlowastlowast

24h48h

72h



0

20

40

60

100

120

8075

Viab

ility

()

C+ Cminus DNA 05 105 1 1 11 1 15 115 1

Chol DOPEC20-18





lowast

lowast


24h48h

72h


0

20

40

60

100

120

8075

Viab

ility

()


C20-20









24h48h

72h





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials





C30-20

0

20

40

60

100

120

8075

Viab

ility

()




lowastlowastlowast

lowast lowast

24h48h

72h



4 Discussion


























5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




















5 Conclusion



Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




Chol DOPE

DC-Chol

EPC

C20-18

C20-20

C30-20



FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials




FP-p

ositi

ve ce

lls (

)

DC-

Chol

DC-

Chol

EPC

C20

-18

C20

-20

EPC

C20

-18

C20

-20

C30

-20

C30

-20

CholDOPE

ns

25

20

15

10

5

0




lowastlowast

lowast

lowast






Acknowledgments



References







































CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials


























CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of


Volume 2014




CoatingsJournal of

Advances in









Volume 2014

MaterialsJournal of

Nano

materials



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