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European Journal of Pharmaceutics and Biopharmaceutics xxx (2014) xxx–xxx

EJPB 11778 No. of Pages 10, Model 5G

18 December 2014

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier .com/locate /e jpb

Research paper

New serine-derived gemini surfactants as gene delivery systems

http://dx.doi.org/10.1016/j.ejpb.2014.12.0130939-6411/� 2014 Published by Elsevier B.V.

⇑ Corresponding author. Department of Life Sciences, Faculty of Sciences andTechnology, University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra,Portugal.

E-mail address: asjurado@bioq.uc.pt (A.S. Jurado).

Please cite this article in press as: A.M. Cardoso et al., New serine-derived gemini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm.http://dx.doi.org/10.1016/j.ejpb.2014.12.013

Ana M. Cardoso a,b, Catarina M. Morais a,b, A. Rita Cruz a,c, Sandra G. Silva d, M. Luísa do Vale d,Eduardo F. Marques d, Maria C. Pedroso de Lima a,b, Amália S. Jurado a,b,⇑a CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugalb Department of Life Sciences, University of Coimbra, Coimbra, Portugalc Faculty of Pharmacy, University of Coimbra, Coimbra, Portugald Centro de Investigação em Química, Department of Chemistry and Biochemistry, University of Porto, Porto, Portugal

a r t i c l e i n f o a b s t r a c t

3031323334353637383940

Article history:Received 7 July 2014Accepted in revised form 8 December 2014Available online xxxx

Keywords:SerineGemini surfactantsTransfection efficiencyPhysico-chemical characteristicsBiodegradable

414243

Gemini surfactants have been extensively used for in vitro gene delivery. Amino acid-derived gemini sur-factants combine the special aggregation properties characteristic of the gemini surfactants with highbiocompatibility and biodegradability. In this work, novel serine-derived gemini surfactants, differingin alkyl chain lengths and in the linker group bridging the spacer to the headgroups (amine, amideand ester), were evaluated for their ability to mediate gene delivery either per se or in combination withhelper lipids. Gemini surfactant-based DNA complexes were characterized in terms of hydrodynamic dia-meter, surface charge, stability in aqueous buffer and ability to protect DNA. Efficient formulations, ableto transfect up to 50% of the cells without causing toxicity, were found at very low surfactant/DNA chargeratios (1/1–2/1). The most efficient complexes presented sizes suitable for intravenous administrationand negative surface charge, a feature known to preclude potentially adverse interactions with serumcomponents. This work brings forward a new family of gemini surfactants with great potential as genedelivery systems.

� 2014 Published by Elsevier B.V.

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1. Introduction

New biocompatible materials, such as gemini surfactants mim-icking natural amphiphilic structures, featured by low toxicity andhigh biodegradability and thus overcoming environmental con-cerns and suiting pharmaceutical applications, have recentlyemerged [17]. Gemini surfactants composed of two sets of an ionicgroup and an hydrocarbon chain, linked by a spacer at the level ofor close to the head group [16], have shown to exhibit special sur-face and aggregation properties, which allow a decrease of the con-centration needed for a therapeutic effect and thus minimize theirpotential toxicity [2,5,22]. The inclusion of aminoacid moieties inthese structures has shown to influence their intermolecularbehavior, due to the chiral centers that allow the formation ofaggregates with a variety of morphologies [4,9,22], and to enhancetheir biodegradability and thus their safe use in biological applica-tions [15,28]. The presence of various functional groups in theamino acid offers the possibility of a great structural variabilityin the resulting surfactants, which can be finely tuned in order to

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obtain an effective control of their aggregation properties and bio-logical activity. This approach may be of utmost relevance in bio-medical applications, for example to modulate pH sensitivity ofsurfactant-containing formulations, through the introduction ofamide/ester bonds, which may be employed in gene delivery topromote DNA cytoplasmic release in response to the low pH ofthe endosomal compartment.

Serine-derived gemini surfactants have been recently synthe-sized for the first time [22]. These surfactants, composed by twoN-alkylated serine amino acid residues, linked to each otherthrough a spacer, bear one positive charge at the amine functionalgroup of each serine residue. This new family of serine-derivedgemini surfactants comprises three different series of compounds,designated, according to the nature of the spacer linkage to theheadgroup, as the amine series (Fig. 1A), the ester and the amideseries (Fig. 1B). For the amine series, compounds with differentalkyl chain lengths were prepared. The impact of this new familyof gemini surfactants on the viability of HeLa cells has been studiedaiming at their application in the pharmaceutical field [21]. Withrespect to their monomeric counterparts as well as to conventionalbis-quaternary gemini surfactants with similar hydrocarbon chainsand spacer length, serine derivatives show the advantage ofexerting lower cytotoxic effects.

(2014),

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In the present work, six members of this new family ofserine-derived gemini surfactants were studied toward their appli-cation as gene delivery systems in gene therapy. For this purpose,serine-derived gemini surfactants were complexed with a reportergene and evaluated for their capacity to mediate transfection,while inducing low cytotoxicity. Complexes were characterizedin terms of physico-chemical properties relevant for their in vivoapplication, such as their hydrodynamic diameter, surface chargeand stability in an aqueous buffer. Furthermore, the ability of thecomplexes to condense and protect DNA was assessed using aDNA-intercalating agent, as an approach to predict the degree ofDNA protection from nuclease degradation. However, in order topromote an efficient gene delivery, the complexes should be ableto dissociate upon interaction with cell membrane structures. Inthis context, membrane-mimicking lipid models were used toevaluate complex cell interactions. In an attempt to unravel theintracellular traffic of the complexes (leading to the endolysosomaldegradative route or bypassing this obstacle), the cellularinternalization pathways used for the uptake of the complexes ableto efficiently mediate transfection were also evaluated.

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2. Materials and methods

2.1. Materials

The gemini surfactants were synthesized by the methodreported by Silva et al. [22] and purified by column chromatogra-phy. The purity of the compounds was assessed by NMR and massspectrometry and further confirmed by the cmc values, obtained by

Fig. 1. Schematic representation of serine-derived gemini surfactants: (A) amineseries, herein designated (nSer)2Nm, (m = 5 and n = 12, 14, 16 and 18); (B) amide,(12Ser)2CON5, and ester, (12Ser)2COO5, series (n = 12). All the surfactants containfully saturated alkyl chains.

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surface tension measurements, which were all in very goodagreement with those already reported [22]. The lipids 1,2-di-oleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol,1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmi-toyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were pur-chased from Avanti Polar Lipids (Alabaster, AL). All the otherchemicals were of the highest grade.

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2.2. Preparation of DNA complexes

Complexes were prepared using the method described by Badeaet al. [3] with a few modifications [6]. Aqueous solutions (0.5 mM)of gemini surfactants were filtered through 0.22 lm pore-diameterfilters (Schleicher & Schuell, BioScience, Germany). Plaincomplexes were prepared by mixing 100 lL of HBS containing0.5 lg of pEGFP-C1 plasmid DNA encoding GFP with aliquots ofthe aqueous gemini surfactant solution, to obtain a gemini/DNAcharge ratios in the range of 1/1–8/1 thereafter incubated at roomtemperature for 15 min. To produce helper lipid/surfactant/DNAcomplexes, aliquots of DOPE:Chol vesicles (see below) were addedto surfactant/DNA complexes to obtain surfactant/helper lipidsmolar ratios in the range of 1/1–1/4, followed by 30 min incubationat room temperature. The DOPE-Chol vesicles were preparedas follows. A solution of 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine (DOPE) and cholesterol (Chol) (Avanti Polar Lipids,Alabaster, AL, USA) in chloroform was dried under vacuum in arotatory evaporator, the resulting lipid films being hydrated withHBS (pH 9.0) to a final lipid concentration of 0.5 mM. The resultingmultilamellar vesicles (MLV) of DOPE plus cholesterol were thensonicated for 3 min and filtered through 0.22 lm pore-diameterfilters (Schleicher & Schuell, BioScience, Germany).

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2.3. Cells

HeLa cells (human epithelial cervical carcinoma cell line) weremaintained in culture at 37 �C, under 5% CO2, in Dulbecco’s Modi-fied Eagle’s medium-high glucose (DMEM-HG; Sigma, St. Louis,MO, USA) supplemented with 10% (v/v) heat inactivated fetalbovine serum (FBS; Sigma, St. Louis, MO, USA), penicillin (100 U/mL) and streptomycin (100 lg/mL). The cells were grown in mono-layer and detached by treatment with 0.25% trypsin solution (Sig-ma, St. Louis, MO, USA).

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2.4. Cell viability

Cell viability was assessed by a modified Alamar Blue assay[24]. This assay takes into account the redox capacity of cells andmeasures the extent of the produced metabolites (resorufin) as aresult of cell growth [13]. Briefly, 48 h after transfection 0.3 mLof 10% (v/v) resazurin dye in complete DMEM-HG medium wasadded to each well. After 45 min of incubation at 37 �C, 150 lL ofthe supernatant was collected from each well and transferred to96-well plates. The absorbance at 570 and 600 nm (informationprovided by the supplier) was measured in a SPECTRAmax PLUS384 spectrophotometer (Molecular Devices, Union City, CA) andcell viability was calculated according to the equation:

Cell viability ð% of controlÞ ¼ ½ðA570 � A600Þ=ðA0570 � A0600Þ� � 100

where A570 and A600 are the absorbances of the samples, and A0570

and A0600 are those of the control (non-treated cells), at the indicat-ed wavelengths.

ini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm. (2014),

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2.5. Transfection efficiency

HeLa cells (0.8 � 105 cell/well) were seeded onto 12-well plates.Following overnight culture, cells were incubated with the differ-ent DNA complexes (0.5 lg of pEGFPC1 per well) at 37 �C under5% CO2 for 4 h, in OptiMEM medium, or in DMEM medium contain-ing 5 or 10% (v/v) FBS. After this period, the medium was replacedwith fresh medium containing 10% (v/v) FBS and antibiotics, andthe cells were further incubated for 44 h to allow gene expression.The transfection efficiency mediated by the different complexeswas evaluated trough analysis of the expression of green fluores-cent protein (GFP) by flow cytometry. Briefly, 48 h after transfec-tion, cells were washed once with PBS and detached with 0.25%trypsin (10 min at 37 �C), and again washed three times by cen-trifugation (950 rpm, 4 �C, 5 min) in ice-cold PBS. The resulting pel-let was resuspended in ice-cold PBS and the samples wereimmediately analyzed. Flow cytometry analysis was performedin live cells using a Becton Dickinson (NJ, USA) FACScalibur flowcytometer. Data were collected and analyzed using CellQuest soft-ware. Live cells were gated by forward/side scattering from a totalof 10,000 events.

2.6. Cellular internalization pathways

To address the mechanisms through which surfactant-basedcomplexes are internalized by cells, recognized inhibitors of differ-ent endocytic pathways were used. Thus, plated HeLa cells washedwith PBS were pre-treated for 30 min, at 37 �C, in serum-free Opti-MEM, with the following endocytosis inhibitors (from Sigma): (i)chlorpromazine (30 lM); (ii) filipin III (5 lg/mL) or (iii) amiloridehydrochloride (5 mM). The cells were then incubated with thecomplexes in the presence of each inhibitor, for 1 h, at 37 �C, inserum-free OptiMEM. To confirm that inhibitors compromiseselectively different endocytic pathways, their effect on the cellularuptake of the fluorescently labeled markers, transferrin, a knownmarker of clathrin-mediated endocytosis, and lactosylceramide, amarker of raft/caveolae-dependent endocytosis, was analyzed.Cytotoxicity was assessed following cell treatment with each ofthe drugs by the Alamar blue assay (see Section 2.4). Flowcytometry analysis was performed as described above (Section 2.5)to evaluate transfection efficiency of the complexes in cellspre-incubated with each of the drugs.

2.7. Surfactant–DNA interactions

Complexes containing 0.2 lg of plasmid DNA, prepared in atotal volume of 100 lL of HBS, were allowed to incubate for further15 min at 37 �C and were then transferred to a 96-well (black-walled) plate (Corning, NY, USA). Hundred microliters of PicoGreen(Molecular Probes, Eugene, OR), diluted according to the manufac-turer’s instructions (1:200 dilution in HBS buffer), were added toeach sample. The fluorescence intensity of PicoGreen, which isdirectly proportional to the amount of accessible/free plasmidDNA, was monitored in a Spex Fluorolog Spectrometer for deter-mining the extent of gemini surfactant–DNA complexation. Theexcitation and emission wavelengths were set at 485 and520 nm, respectively. The degree of plasmid DNA protection con-ferred by the complexes, taken as proportional to the surfactant/DNA complexation, was calculated as follows:

PDNA ¼ 1� ½ðF � F100Þ=ðF0 � F100Þ�

where F is the fluorescence intensity measured after adding thePicoGreen solution to the complexes, F0 (corresponding to 0% ofplasmid DNA protection) was obtained by using free plasmid DNAin the same amount as that associated with the complexes,F100 (corresponding to 100% of plasmid DNA protection) is the

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residual fluorescence intensity of a negative control obtained byusing a PicoGreen solution without plasmid DNA, which mimickeda situation of 0% of DNA available to the PicoGreen solution.

Destabilization of the complexes was accomplished by incubat-ing them for 15 min with small unilamellar vesicles (SUV) of DOPCor of a mixture of DOPE:DOPC:POPS (2:1:1 molar ratio) andevaluated through PicoGreen fluorescence intensity as the differ-ence between the protection obtained for complexes in theabsence of the lipid vesicles and that obtained in their presence.

2.8. Physico-chemical properties of the DNA complexes (particle size,zeta potential, colloidal stability)

The size of the surfactant-based structures was assessed using aSubmicron Particle Size Analyzer, Beckman Coulter N4 Plus. Thecolloidal suspensions of the complexes were diluted with HBS,and the particle size analysis was carried out at a scattering angleof 90� and a temperature of 25 �C.

The Zeta potential of the surfactant-based structures was mea-sured using a Zeta sizer Nano ZS, ZN 3500, with a 532 nm laser(Malvern Instruments, UK). The measurements were performedin HBS, at 25 �C, using DTS 1060C disposable Zeta cells and the pro-tocol for general purposes (medium viscosity 0.89 cP, mediumrefractive index 1.33, sample viscosity 0.89 cP, particle refractiveindex 1.45 and equilibration time 3 min). Values of dielectric con-stant of 78.5 and beam mode F(Ka) of 1.5 (Smoluchowsky) wereused for Zeta potential determination.

The turbidity of gemini surfactants and complex dispersions,providing a measure of their colloidal stability, was monitored asa function of time on a SPECTRAmax PLUS 384 spectrophotometer(Molecular Devices, Union City, CA) at a wavelength of 550 nm. Thefinal concentration of lipid plus gemini surfactant was 0.1 mM inHBS buffer, pH 7.4. The turbidity values were registered over aperiod of 48 h.

2.9. Statistical analysis

Data are presented as mean ± SD. The significance of the resultswas statistically analyzed by a one-way analysis of variance(ANOVA) with Tukey’s multiple pairwise comparison. Statisticalsignificance was set at p < 0.05.

3. Results

3.1. Biological activity of serine-derived gemini surfactant-based DNAcomplexes

At cellular level, an efficient nonviral gene delivery system hasto encapsulate/complex the nucleic acids and confer protectionfrom degradation by intracellular and extracellular nucleases, carrythem into the cell, release its cargo next to the intracellular targetand promote high gene expression, without causing cytotoxicity. Inthis regard, cytotoxicity of the plain (gemini surfactant/DNA) andternary (gemini surfactant/DNA/helper lipids) complexes wasevaluated, and those complexes that did not promote significantloss of cell viability were selected for evaluating their ability tomediate transfection. The internalization pathways used by thecomplexes able to efficiently mediate gene expression were alsoassessed.

3.1.1. Cytotoxicity induced by serine-derived gemini surfactant-basedDNA complexes

Fig. 2 shows the results obtained for the toxicity of surfactant-based DNA complexes in HeLa cells. As observed, the cytotoxiceffect was dependent on the (±) charge ratio of the complexes,

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Fig. 2. Viability of HeLa cells upon incubation with complexes composed of thesurfactant (indicated in the figure), without (h) or with (j) the helper lipids DOPEand cholesterol (2:1 molar ratio), at the indicated (±) charge ratios. Cell viabilitywas expressed as a percentage of a control (non-treated cells). Pairwise datacomparisons were performed for the helper lipids-containing complexes vs. thecorresponding helper lipids-free complexes at the same (±) charge ratio (⁄p < 0.05,⁄⁄p < 0.01).

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increasing for the highest charge ratios tested. The complexes thatinduced the highest loss of cell viability were those formulatedwith (12Ser)2CON5 and (18Ser)2N5 at the 8/1 (±) charge ratio, bothin the absence and presence of helper lipids. On the other hand,complexes prepared with (12Ser)2COO5 and (16Ser)2N5 were theleast toxic, particularly at the lowest charge ratios assayed. Fur-thermore, at the highest charge ratio, cytotoxicity was found todecrease with increasing hydrocarbon chain length of the surfac-tant, for alkyl chains from 12 to 16 carbon atoms-long. However,this trend was reverted by (18Ser)2N5-based complexes, whichinduced the highest cytotoxicity among the complexes containingsurfactants of the amine series. Concerning the effect of the natureof the spacer linkage on cell viability, comparison of the analogouscompounds of each series shows that complexes containing surfac-tants of the ester series are the least cytotoxic, followed by com-plexes containing surfactants of the amine series and at lastthose with surfactants of the amide series.

3.1.2. Transfection efficiency of serine-derived gemini surfactant-basedDNA complexes

Complexes that did not cause significant toxicity to HeLa cellswere selected for the evaluation of their ability to mediate genedelivery. As observed in Fig. 3A, complexes prepared in the absenceof helper lipids lack the ability to efficiently mediate gene expres-sion. The only exception was found for complexes composed of(18Ser)2N5 surfactant prepared at the 2/1 (±) charge ratio, which

Fig. 3. Transfection efficiency of gemini surfactant-based DNA complexes formu-lated without (h) or with (j) the helper lipids DOPE and cholesterol (2:1 molarratio), at the indicated (±) charge ratios (A) and of (12Ser)2COO5:HL-based DNAcomplexes prepared at the 3/2 ( ) and 2/1 (j) (±) charge ratios, performed in theabsence (0%) or presence of increasing amounts of serum (5% and 10%) (B). Datacomparisons were performed for helper lipid-containing complexes vs. the corre-sponding helper lipid-free complexes at the same (±) charge ratios (⁄p < 0.05,⁄⁄p < 0.005, ⁄⁄⁄p < 0.001).

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were able to transfect 21% and 32% of cells in the presence andabsence of helper lipids, respectively, albeit no significant differ-ence was obtained between the two formulations. Although thegemini surfactants of each series (amine, amide and ester) with12 carbon alkyl chains (C12) were unable to efficiently mediatetransfection per se, the corresponding complexes formulated withthe helper lipids DOPE and cholesterol presented significant trans-fection efficiency (up to 27%, 37% and 50%, respectively for(12Ser)2CON5, (12Ser)2N5 and (12Ser)2COO5). Interestingly, thehighest levels of transfection were found for the lowest chargeratios tested (1/1–2/1). Comparison with the commercial reagentLipofectamine 2000 showed that, although this reagent was ableto transfect more cells than the serine-derived gemini surfactant-based complexes (74%, data not shown), it induced significantlyhigher levels of cytotoxicity (only 35.6% of viable cells, data notshown).

Transfection experiments in the presence of increasing amountsof serum were performed using the formulations containing theester, the amide and the amine gemini surfactants with C12 tailsand helper lipids. As observed in Fig. 3B, no statistically significantdifferences in the transfection efficiency of complexes containingthe ester-derived gemini surfactant, prepared at the 3/2 and 2/1(±) charge ratios, were found in the absence or presence of serum(up to 10%).

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3.1.3. Cellular internalization pathways used for the uptake of serine-derived gemini surfactant-based DNA complexes

The mechanisms involved in the internalization of serine-derived gemini surfactant-based DNA complexes by HeLa cellswere studied for the complexes that presented the highest trans-fection ability. Cells were treated with drugs that selectively inhibitendocytic processes that occur at different membrane domains,namely clathrin-mediated endocytosis, which takes place in non-lipid raft domains, macropinocytosis, which occurs in mixed raftand non-raft domains, and raft-mediated endocytosis, whichincludes caveolae-mediated endocytosis [8]. Cellular internaliza-tion mechanisms were identified by flow cytometry, through theevaluation of transfection efficiency of cells pre-incubated withthe pharmacological inhibitors of clathrin-mediated endocytosis(chlorpromazine), macropinocytosis (amiloride hydrochloride)and endocytic processes occurring in lipid raft domains of the cellmembrane (filipin III).

The complexes containing surfactants with 12 carbon alkylchains of each of the three series and helper lipids showed to bepreferentially internalized by macropinocytosis, since the lowestlevel of transfection was observed upon treatment of cells withamiloride hydrochloride (Fig. 4). Among these complexes, thoseprepared with (12Ser)2CON5 and (12Ser)2COO5 surfactants werealso internalized through cholesterol-enriched membrane domains(lipid-rafts), as concluded from the significant reduction in trans-fection observed in filipin III-treated cells. The clathrin-mediatedendocytosis showed to have also a significant contribution to inter-nalize complexes prepared with (12Ser)2N5 and (12Ser)2COO5.Complexes prepared with (18Ser)2N5 in the absence and presenceof helper lipids were compared in terms of the cellular internaliza-tion pathways. While the complexes devoid of helper lipids werenot internalized by any of the endocytic pathways evaluated, sug-gesting the capacity to cross membranes through direct interactionwith membrane lipids, the complexes formulated with helperlipids were partly taken up by a clathrin-mediated endocytosisprocess and partly by macropinocytosis.

Noteworthy, the transfection efficiency observed for all thestudied complexes upon cell treatment with endocytosis inhibitorsdid not decay to residual values, suggesting that a membrane lipid-mediated process may be involved in their cell uptake.

ini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm. (2014),

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Fig. 4. Effect of different endocytosis inhibitors on transfection mediated bysurfactant-based DNA complexes. HeLa cells were incubated with either 30 lM ofchlorpromazine (h), 5 lg/mL of filipin III ( ), or 2.5 mM of amiloride hydrochloride(j) and transfected with the complexes indicated in the figure. Transfection in thepresence of each inhibitor was expressed as percentage of transfected cells witheach complex but not treated with the inhibitors of internalization pathways. Dataare presented as mean ± standard deviation, representative of at least threeindependent experiments. Data comparisons were performed between eachcondition tested and the respective control corresponding to 100% of transfectedcells (⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001).

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3.2. Characterization of serine-derived gemini surfactant/DNAcomplexes

Interaction of gemini surfactants with opposing charged DNA isregarded as a key feature for the efficient nucleic acid com-plexation and protection from nuclease degradation. As other non-viral gene delivery systems, gemini surfactant-based DNAcomplexes should meet some criteria, in terms of surface charge,hydrodynamic diameter and stability, toward successful transfec-tion. In this regard, complexes displaying high transfection effi-ciency as well as those unable to transfect cells were evaluatedfor those parameters in order to ascertain the physico-chemicalproperties responsible for efficiency in gene delivery.

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3.2.1. Protection of DNA by serine-derived gemini surfactantsThe ability of gemini surfactants to interact and protect DNA

was evaluated using the fluorescent probe PicoGreen, whichintercalates nucleic acid double strands and whose fluorescenceis dramatically enhanced in this condition. Fig. 5 displays thepercentage of DNA inaccessible to the probe, expressed in termsof DNA protection conferred by the gemini surfactants or thegemini surfactants plus the helper lipids DOPE and cholesterol. Ahigher degree of DNA protection was observed with increasing

Fig. 5. Gemini–DNA interactions, as assessed by PicoGreen accessibility to DNA complexeand Cholesterol (2:1 molar ratio) (B), at the indicated (±) charge ratios. The maximum fluwere performed for the complexes at different (±) charge ratios vs. the same complex⁄⁄⁄p < 0.001).

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(±) charge ratio of the complexes, formulated both in the absenceand presence of helper lipids. The ester-derived surfactant showedthe lowest levels of protection (Fig. 5A), displaying high protectionability only at the highest (±) charge ratio tested (8/1). Protectionpromoted by (14Ser)2N5 and (18Ser)2N5 was very efficient evenat the (±) charge ratio of 2/1 (more than 80%) in contrast to(12Ser)2N5, which showed high protection only at the (±) chargeratio of 4/1 or higher, and to (16Ser)2N5, which exhibited muchlower protection levels at all the (±) charge ratios assayed. There-fore, no direct relationship between alkyl chain length and efficien-cy of DNA protection was found. The addition of helper lipids to thecomplexes decreased the degree of DNA protection conferred bythe surfactants at the highest charge ratios, except in the case ofthe complexes containing (14Ser)2N5 and (18Ser)2N5, whichshowed similar abilities to protect DNA in the absence and pres-ence of helper lipids over the range of charge ratios tested. Onthe other hand, at the lowest charge ratios, (12Ser)2COO5 and(12Ser)2N5 complexes exhibited higher capacity to avoid DNA–probe interactions in the presence of helper lipids. In contrast,complexes of (16Ser)2N5 and helper lipids conferred lower protec-tion to DNA than the corresponding complexes composed of neatsurfactant at all the charge ratios tested.

3.2.2. Physico-chemical properties of surfactant-based DNA complexesThe size of the complexes was evaluated by dynamic light scat-

tering and was found to vary depending on composition andcharge ratio (Table 1). The sizes of the complexes were found torange from 163 nm to more than 3 lm, all samples showing a poly-dispersity index higher than 0.3, and being therefore analyzed aspolydisperse samples. Complexes formulated with the surfactantshaving long hydrocarbon chains (16 and 18 carbon atoms) showedat all (±) charge ratios an increase in size promoted by the additionof the helper lipids. The complexes formulated with surfactantscontaining short hydrocarbon chains (C12 and C14) showed thesame tendency, except at the lowest (±) charge ratio assayed (1/1for the complexes of surfactants C12 and 2/1 for the (14Ser)2N5-based complexes), under which condition the helper lipids induceda decrease in size.

The surface charge of the complexes composed of gemini sur-factant/DNA and gemini surfactant/DNA/helper lipids was assessedby Zeta potential measurements (Table 2). The surface charge ofthe structures formed by gemini surfactants and gemini surfac-tants plus helper lipids in aqueous suspensions, in the absence ofDNA, was also measured for comparison. As observed in Table 2,the particles composed of surfactants exhibited a positive surface

d with gemini surfactants (A) or gemini surfactants plus the helper lipids (HL) DOPEorescence (control) corresponds to maximum DNA accessibility. Data comparisonsformulations at the immediately precedent (±) charge ratio (⁄p < 0.05, ⁄⁄p < 0.01,

ini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm. (2014),

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Table 1Hydrodynamic diameter of DNA complexes formulated with gemini surfactants at different (±) charge ratios, in the absence and in the presence of the helper lipids DOPE andcholesterol.

Hydrodynamic diameter (nm)a

1/1 3/2 2/1 4/1 8/1

(12Ser)2CON5 970 ± 140 1740 ± 190 1662 ± 467 560 ± 140 290 ± 27(12Ser)2CON5:DOPE:Chol 940 ± 100 >3000 2221 ± 674 1032 ± 43 460 ± 45

(12Ser)2COO5 490 ± 200 271 ± 63 393 ± 41 355 ± 41 2000 ± 348(12Ser)2COO5:DOPE:Chol 387 ± 13 410 ± 14 454 ± 12 960 ± 140 2413 ± 266

(12Ser)2N5 820 ± 220 1232 ± 114 2239 ± 629 526 ± 65 267 ± 13(12Ser)2N5:DOPE:Chol 800 ± 170 2827 ± 376 2747 ± 362 1022 ± 95 500 ± 250

(14Ser)2N5 – – 2389 ± 692 312 ± 43 163 ± 9(14Ser)2N5:DOPE:Chol – – 1624 ± 399 1582 ± 151 919 ± 36

(16Ser)2N5 – – 233 ± 25 497 ± 85 1374 ± 102(16Ser)2N5:DOPE:Chol – – 607 ± 42 780 ± 190 1824 ± 399

(18Ser)2N5 – – 850 ± 120 196 ± 14 166 ± 9(18Ser)2N5:DOPE:Chol – – 2527 ± 478 304 ± 31 222 ± 10

a Results represent mean ± SD of at least three independent experiments. All samples were polydisperse.

Table 2Zeta potential of DNA complexes formulated with gemini surfactants at different (±) charge ratios, in the absence or in the presence of the helper lipids DOPE and cholesterol.

f-Potential (mV)a

1/1 3/2 2/1 4/1 8/1 No DNA

(12Ser)2CON5 �14 ± 11 �5 ± 12 +18 ± 8 +18 ± 6 +14 ± 5 +6 ± 8(12Ser)2CON5:DOPE:Chol +21 ± 8 +28 ± 7 +30 ± 9 +36 ± 8 +32 ± 10 +30 ± 8

(12Ser)2COO5 �16 ± 11 �26 ± 7 �27 ± 8 �23 ± 10 �10 ± 10 +2 ± 9(12Ser)2COO5:DOPE:Chol �35 ± 7 �38 ± 7 �37 ± 7 �21 ± 10 +4 ± 13 +14 ± 8

(12Ser)2N5 �21 ± 8 �23 ± 9 �21 ± 7 �7 ± 14 �3 ± 11 +17 ± 9(12Ser)2N5:DOPE:Chol �38 ± 7 �28 ± 8 �13 ± 10 +13 ± 7 +19 ± 5 +17 ± 9

(14Ser)2N5 – – +13 ± 10 +9 ± 3 +13 ± 7 +6 ± 2(14Ser)2N5:DOPE:Chol – – +27 ± 2 +25 ± 3 �35 ± 2 �40 ± 3

(16Ser)2N5 – – +22 ± 5 +31 ± 2 +23 ± 2 +52 ± 9(16Ser)2N5:DOPE:Chol – – +18 ± 2 +16 ± 1 �39 ± 2 �42 ± 3

(18Ser)2N5 – – +31 ± 3 +29 ± 5 +34 ± 4 +46 ± 5(18Ser)2N5:DOPE:Chol – – �35 ± 4 �43 ± 2 �44 ± 2 �49 ± 3

a Results represent mean ± SD of at least three independent experiments. All samples were monodisperse.

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charge. The complexation of DNA by the surfactants containingC12 alkyl chains led to a change in the Zeta potential to negativevalues, although for complexes of (12Ser)2CON5 the signal inver-sion occurred only at the lowest (±) charge ratios assayed (1/1and 3/2). Surfactants with longer hydrocarbon chains maintainedthe positive Zeta potential upon complexation with DNA, at leastat (±) charge ratios higher or equal to 2/1. In the absence of DNA,the addition of helper lipids to the surfactants containing 12 car-bon alkyl chains did not alter the sign of the surface charge. How-ever, for surfactants with longer hydrocarbon chains, the helperlipids inverted the Zeta potential value, from positive to negative.Complexes of (12Ser)2CON5 prepared with helper lipids showeda highly positive charge over the range of (±) charge ratios tested.The incorporation of helper lipids into the complexes of surfactants(12Ser)2N5 and (12Ser)2COO5 produced structures with positiveZeta potential at high surfactant/DNA charge ratio and with nega-tive Zeta potential at low surfactant/DNA charge ratio. The oppo-site occurred for complexes of surfactants containing longerhydrocarbon chains and formulated with helper lipids. Complexesof (14Ser)2N5 and (16Ser)2N5 displayed negative Zeta potential atthe highest (±) charge ratio (8/1) and positive surface charge at theother (±) charge ratios. Furthermore, (18Ser)2N5-containing com-plexes formulated with helper lipids presented a highly negativeZeta potential in the range of charge ratios assayed.

Evaluation of the colloidal stability of the surfactant-based DNAcomplexes, by monitoring the absorbance at 550 nm over a period

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of 48 h, revealed that all complexes were extremely stable over aperiod of at least 24 h (data not shown). This aspect is particularlyimportant for intravenous administration of freshly prepared com-plexes, in order to ensure their prolonged blood circulation.

3.3. Interaction of surfactant/DNA complexes with lipid membranemodels

Destabilization of DNA complexes upon interaction with cellu-lar membranes is essential for nucleic acid release in or close totheir intracellular target, in order to allow gene expression.

Destabilization of the serine-derived gemini surfactant-basedDNA complexes was evaluated upon interaction with lipid vesicleswith different composition (neutral DOPC vesicles, and negativelycharged DOPE:DOPC:POPS (2:1:1) vesicles). Fig. 6 shows the effectsexerted by the two lipid membrane models on complex stability,reflected by the exposure of the DNA to the fluorescent probe(PicoGreen). Complexes prepared at high (±) charge ratios weremore extensively destabilized than those prepared at lower chargeratios. Among the complexes formulated with surfactants contain-ing 12 carbon alkyl chains, those possessing an ester bond present-ed the lowest level of destabilization and their stability did notshow to be affected by the presence of helper lipids. In contrast,the addition of helper lipids to complexes formulated with(12Ser)2N5 and (12Ser)2CON5 surfactants led to a decrease in thesusceptibility to destabilization induced by both types of mem-

ini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm. (2014),

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Fig. 6. Destabilization of surfactant-based complexes upon interaction with lipidvesicles of DOPC (A) or of a mixture of DOPE:DOPC:POPS (2:1:1 molar ratio) (B),evaluated in terms of PicoGreen (fluorescent probe) access to DNA. The surfactant/DNA (white bars) or surfactant/DNA/helper lipids (black bars) complexes, at theindicated (±) charge ratios, were incubated with a PicoGreen solution (dilution1:200) and lipid vesicles were then added in a two-fold excess over the surfactantconcentration. Data are presented as percentage of maximum complex destabi-lization obtained with a control prepared with PicoGreen and free DNA (in the sameamount as that associated with the complexes), corresponding to maximumPicoGreen accessibility to DNA. The results represent the mean ± standard deviationobtained from three independent experiments. Data comparisons were performedbetween formulations prepared with and without helper lipids (⁄p < 0.05,⁄⁄p < 0.005, ⁄⁄⁄p < 0.001).

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branes. Differences in the destabilizing effects exerted by the zwit-terionic and the anionic vesicles were noticeable for complexesformulated with surfactants containing hydrocarbon chains with14 or more carbon atoms. Whereas complexes containingsurfactants with 14 and 16 carbon alkyl chains were less destabi-lized by anionic lipid vesicles than by the zwitterionic vesicles,complexes formulated with (18Ser)2N5 were not destabilized byDOPC vesicles but presented a high degree of destabilization inthe presence of the anionic lipid mixture, especially thosecomplexes containing helper lipids.

4. Discussion

A systematic comparative study of the ability of six members ofa new family of serine-derived gemini surfactants to safely medi-ate gene delivery and transfection has been conducted in the pre-sent work. The gemini surfactants of this family were selected inorder to examine the influence of (i) the length of the hydrocarbonchains (C12, C14, C16 and C18), specifically in the amine series, and(ii) the nature of the linkage between the cationic headgroup andthe spacer (amine, amide and ester) on the transfection efficiency.For this purpose, plain gemini surfactant/DNA complexes and tern-ary complexes of gemini surfactant/DNA/helper lipids, resultingfrom the addition of DOPE and cholesterol liposomes (2/1 molarratio) to plain complexes, were evaluated as safe gene delivery sys-tems and characterized in terms of their physico-chemical proper-ties. Destabilization of the gemini surfactant-based complexes wasmonitored upon the addition of membrane mimicking lipidvesicles in order to shed light on the importance of complex–membrane interactions in their ability to mediate gene delivery.For the sake of clarity, these data will be discussed from two per-

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spectives: (a) the influence of the hydrocarbon chain length and(b) the importance of the nature of the headgroup/spacer linkagein the biological activity of serine-derived gemini surfactants.

4.1. Influence of the hydrocarbon chain length on the biologicalactivity of serine-derived gemini surfactants

Concerning the surfactants of the amine series (effect of alkylchain length), in the presence of helper lipids, all the surfactantsstudied were able to mediate gene delivery to some extent. How-ever, no clear trend of the effect of alkyl chain length on transfec-tion efficiency was observed. The surfactant containing the 16carbon alkyl chain, (16Ser)2N5, was found to be the least efficientof the series. This observation seems to contrast with that foundfor complexes of conventional bis-quaternary gemini surfactants,for which the surfactant containing 16 carbon alkyl chainsexhibited the highest transfection efficiency [6]. However, caremust be taken before a direct comparison is made. The differentbehavior of the conventional vs. the serine-derived gemini surfac-tants in terms of transfection can be assigned either to the natureof their headgroups (simple alkyl ammonium vs. serine-derivedammonium), or to the length of the spacers (2 methylene vs. 5methylene groups). In fact, the volume occupied by the cationicheadgroup of serine-derived gemini surfactants is much largerthan that of the conventional bis-quat gemini surfactants,whose headgroups are composed of simple dimethyl quaternaryammonium groups instead of the larger serine residues. On theother hand, the spacer length is important in governing geminisurfactant interactions with other molecules. According to Wettiget al. [27], surfactant ammonium groups should be 4.9 Å apart inorder to better interact with adjacent phosphate groups of DNA.This distance has been reported to be equivalent to the trimethy-lene spacing in spermine, which is closest to that determined forgemini surfactants containing a two- [12] to three-methylenespacer [27]. However, the presence of large headgroups, such asthose of the serine-derived gemini surfactants, can result in anefficient accommodation of the DNA phosphate groups due tothe flexibility of the surfactant chemical structure, namely at thelevel of the number of rotatable C–C bonds [12].

Curiously, DNA protection provided by the complexes preparedwith serine-derived gemini surfactants with different hydrocarbonchain lengths followed a similar variation to that observed for thetransfection efficiency, i.e. the (16Ser)2N5-based complexes werethose that conferred the lowest protection to DNA and exhibitedthe lowest transfection efficiency. Interestingly, these complexesshowed also the lowest cytotoxicity. Taking into considerationthe effects on cytotoxicity and DNA protection exerted by plaincomplexes prepared with gemini surfactants containing 14, 16and 18 carbon alkyl chains at the 8/1 (±) charge ratio, the followingsequence (18Ser)2N5 P (14Ser)2N5 > (16Ser)2N5 can be estab-lished. This turning point has been already found in terms of thecytotoxicity of this series of serine-derived surfactants [21], anevent that we interpreted in the light of the cutoff-effect describedfor the interaction of alcohols of increasing chain length with lipidmembranes [19]. However, the cytotoxicity of the surfactants perse was found to vary with the hydrocarbon chain length inverselyto the corresponding complexes, i.e. (16Ser)2N5 > (14Ser)2N5 >(18Ser)2N5 [21].

In order to improve the transfection efficiency of serine-derivedgemini surfactants, the helper lipids DOPE and cholesterol wereused in combination. The helper lipid DOPE confers the systemthe ability to escape lysosomal degradation, while cholesterolwas found to be a more biocompatible lipid and less prone tointeract with serum proteins [10,14,25]. The addition of helperlipids to complexes containing serine-derived gemini surfactantswith 14–18 carbon atom alkyl chains increased their cytotoxicity.

ini surfactants as gene delivery systems, Eur. J. Pharm. Biopharm. (2014),

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Apparently, helper lipid-induced cytotoxicity had only a negativeimpact on the transfection efficiency in the case of complexesprepared with (18Ser)2N5. Despite the increase of cytotoxicity,the addition of helper lipids promoted increased transfection levelsfor complexes containing (14Ser)2N5 and did not significantlyaffect transfection induced by complexes containing (16Ser)2N5.In view of the disparity of effects exerted by helper lipids on thetransfection efficiency of complexes containing gemini surfactantswith hydrocarbon chains of different length, it is expected thatthose lipids induce the formation of structurally different aggre-gates, depending on the incorporated surfactants. In fact, ourresults of complex surface charge showed that the Zeta potentialvalues of (18Ser)2N5-based complexes change from positive tonegative upon addition of helper lipids, which presented negativesurface charge per se, indicating that the interaction with thepre-formed complexes is mostly at the surface level. Thus, theDOPE:Chol vesicles should remain surface-exposed and a stronggemini–DNA interaction, most likely both electrostatic andhydrophobic, would prevent major internal rearrangements inthe ternary complexes. Complexes containing the (16Ser)2N5 and(14Ser)2N5 gemini surfactants showed also an inversion of the Zetapotential, from positive to negative, upon addition of helper lipids,albeit only at the highest (±) charge ratio (8/1). On the other hand,complexes containing gemini surfactants with the shortest chains(C12), both from amine and ester series, presented negative Zetapotential values, in the range of (±) charge ratios assayed, suggest-ing a lower capacity of those surfactants to hide the negativelycharged phosphate groups from DNA. For these complexes, theaddition of helper lipids did not significantly affect the Zeta poten-tial, except at the highest (±) charge ratios, at which an inversion ofZeta potential to positive values occurred. Apparently, in this case,the helper lipids promoted a structural rearrangement leading tothe exposure of the surfactant positive charges. In terms of DNAprotection this structural rearrangement induced by helper lipidsled to a significant decrease of the protection afforded by (12Ser)2-

N5 and (12Ser)2COO5 to the DNA. The (14Ser)2N5 and (18Ser)2N5-based complexes conferred high levels of protection both in theabsence and presence of helper lipids. For (16Ser)2N5-based com-plexes the relatively low protection offered by the plain geminicomplex was not improved by the addition of helper lipids. Thelack of impact of helper lipids on DNA protection afforded by thecomplexes based on long-chain amine derivatives seems to berelated to an apparent stability of complex microstructure core,where DNA is hidden in (14Ser)2N5 and (18Ser)2N5-basedcomplexes or relatively accessible to PicoGreen probe in (16Ser)2

N5-based complexes.Considering now the mixtures of gemini surfactant plus helper

lipids without DNA complexation (no DNA, in Table 2), it is inter-esting to note that, whereas for surfactants displaying chains with14 carbon atoms or more the addition of helper lipids promoted analteration of the Zeta potential from positive to negative, for thesurfactants with the shortest chains (C12) of the different series(amine, ester and amide), helper lipids did not alter (amine series)or promoted an increase of the Zeta potential positive values (esterand amide series). Unfortunately, we do not have data to allow usto infer the nature of the interactions between gemini surfactantsand the lipid vesicles. The interesting study of Akbar et al. [1]demonstrated an antagonistic mixing between m–s–m gemini sur-factants containing C16 chains and different spacers and the zwit-terionic lipid DOPE. The authors established a clear dependence ofthe magnitude of the antagonistic behavior on the critical packingparameter (CPP) of the corresponding amphiphiles in the gemini/DOPE system, the weakest antagonistic interaction being detectedfor the gemini surfactant (16-12-16) exhibiting the closest CPP val-ue to that of DOPE. Although our lipid system is different from thatof Akbar et al., consisting of vesicles of DOPE and cholesterol, the

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length of the chains of the surfactants used in the present study(C12 vs. C14 to C18) is expected to influence CPP compatibility inthe mixture with DOPE/cholesterol vesicles. This fact shouldexplain the difference between the Zeta potential variation causedby the addition of helper lipids, changing from positive to negativeonly in the case of surfactants containing C14 or longer chains.

Regarding the interaction of the complexes with lipid vesicles, itis interesting to observe that the plain complexes containing thesurfactants with the shortest hydrocarbon chains were moreextensively destabilized than those containing surfactants withlonger hydrocarbon chains, and that only the (18Ser)2N5 surfac-tant-containing complexes were preferentially destabilized byanionic lipid vesicles. Curiously, the addition of helper lipids to(18Ser)2N5-containing complexes significantly increased theirdestabilization, specially by the anionic lipid vesicles, in contrastto what occurred with the other complexes. The negative impactthat helper lipids exhibited on the transfection efficiency of(18Ser)2N5-based complexes may be attributed not only to theincreased cytotoxicity, but also to a detrimental lack of DNA pro-tection upon interaction with membranes.

4.2. Importance of the nature of the headgroup/spacer linkage in thebiological activity of serine-derived gemini surfactants

Regarding the influence of the nature of the headgroup/spacerlinkage on the biological behavior of gemini surfactants, it has beenshown that the incorporation of chemical bonds susceptible ofbeing enzymatically cleaved, such as ester and amide, in the mole-cular structure of delivery system components, reduces their cyto-toxicity and enhances their ability to produce efficient complexeswith DNA [11]. Consistent with this finding, data obtained withcomplexes prepared with surfactants of each of the three series(amine, amide and ester) possessing C12 alkyl chains showedlow cytotoxicity and high transfection efficiency, although the lat-ter was observed only when complexes were formulated withhelper lipids. In HeLa cells, the lowest cytotoxicity and the highesttransfection efficiency were displayed by complexes containing thesurfactant of the ester series.

It is worth noting that the effective length of the spacer for thegemini surfactants of the ester and amide series, i.e. the distanceseparating the two quaternary ammonium groups, correspondsto 11 methylene groups, because the linkage of the spacer to theserine residue is made through the a-carboxylate group of the ami-noacid, rather than directly to the charged nitrogen atom. There-fore, these gemini surfactants should have the ability to coil theirflexible spacer in order to place the charged headgroups at theideal distance to interact with the phosphate groups of DNAmolecules [12]. This ability to refine the interaction with DNAmolecules is likely higher in the ester series, since the C–N bondof the amide linkage, in contrast to the C–O bond of the ester link-age, has a high double bond character that constrains free rotationof this bond, thus reducing the flexibility of the spacer. Notewor-thy, in human embryonic kidney cells (Hek293), complexes con-taining the amide derivative and helper lipids showed thehighest transfection ability (transfecting 32% of the cells at the 2/1 (±) charge ratio), whereas complexes formulated with the esterderivative and helper lipids were able to efficiently transfect 19%of the cells at the same charge ratio. No significant toxicity wasobserved in these cells for both types of complexes. This observa-tion clearly indicates that other factors, besides those concerningthe characteristics of vector components and their interactionswith DNA, should be involved in transfection efficiency ofsurfactant-based delivery systems, namely cell-specific features,such as cytoplasmic membrane composition and mechanisms ofcomplex internalization, thus justifying that the same complexesshowed different competence to transfect HeLa and Hek293 cells.

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Considering the plain complexes (without helper lipids), it isprobable that the interaction of the least flexible surfactantmolecules (amine and amide derivatives) with DNA originatesmore steric hindrance with consequent higher protection levels,as compared to those afforded by the surfactant of the ester series.However, the transfection efficiency of the complexes preparedwith gemini surfactants and helper lipids does not seem to berelated with the DNA protection afforded by these components.For those formulations able to efficiently mediate transfection, acompromise should exist between the ability of complexes toprotect DNA and to release it inside the cell. The eventual intracel-lular degradation of the labile amide and ester bonds may lead tothe disaggregation of the complexes and consequent nucleicacid release. In fact, although the complexes incorporating(12Ser)2COO5, containing or not helper lipids, showed to be lessefficient in releasing DNA upon interaction with membranes, ascompared to complexes prepared with the amide, (12Ser)2CON5,or amine, (12Ser)2N5, homologues, they presented the highesttransfection efficiency. Thus, DNA release should occur inside thecell, where acidification of the endosomal compartment could trig-ger structural changes in the surfactant molecules (e.g. hydrolysisof the ester bond), leading to complex destabilization and nucleicacid release into the cytoplasm.

Regardless of what has been described for lipoplexes about therelationship between size and transfection efficiency [23], largecomplexes containing the gemini surfactants were found not tobe those presenting the highest transfection ability. In fact, thecomplexes that induced the highest transfection levels (formedby the ester derivative, DNA and helper lipids at low (±) chargeratios) displayed rather small sizes (387–454 nm). The small sizesof the complexes are important for intravenous administration tocircumvent capillary retention. Previous studies of our group [6]with DNA-complexes based on conventional (m–s–m) gemini-surfactants, prepared by the same methodology used in thepresent work to obtain the complexes based on serine-derivedcomplexes, allow us to establish a rough comparison betweenthe hydrodynamic diameter of these two types of surfactant-basedcomplexes. Taking into account the length of the alkyl chains andof the spacer, this comparison can be established between(12Ser)2N5-containing complexes and complexes prepared with12-5-12 and between complexes based on (12Ser)2CON5 or(12Ser)2COO5 and complexes prepared with 12-10-12, since thedistance between the two headgroups in surfactants presentingan ester or amide headgroup-spacer linkage is equivalent to ca.11 methylene groups. We observe that complexes based onserine-derived gemini surfactants are generally smaller than thosebased on conventional gemini surfactants. The only exceptionconsists of the (12Ser)2N5-based complexes prepared with helperlipids at the 8/1 (±) charge ratio, which are slightly larger thanthe corresponding 12-5-12-based complexes. Complexes basedon the ester and amide serine derivatives are all smaller than12-10-12-containing complex counterparts, with the only excep-tion of complexes prepared with the ester-derived gemini surfac-tant and helper lipids at the 8/1 (±) charge ratio. Additionally, itis worth noticing that the complexes prepared with the serinederivatives display a sharper size distribution than those preparedwith conventional gemini surfactants, indicating that, in contrastto the latter, they exist in a relatively homogeneous single popula-tion. Unfortunately, 12-5-12 and 12-10-12 surfactants producedcomplexes which, besides exhibiting very low transfectionefficiency, revealed to be highly cytotoxic, even when formulatedwith helper lipids, thus being unsuitable for gene deliverypurposes [6]. This fact prevented us to pursue a more extensivecomparison between conventional gemini surfactant- vs.serine-derived gemini surfactant-based complexes regarding theirhydrodynamic sizes and efficiency in transfection.

Please cite this article in press as: A.M. Cardoso et al., New serine-derived gemhttp://dx.doi.org/10.1016/j.ejpb.2014.12.013

Noteworthy, the hydrodynamic diameter of the most efficientcomplexes described here is similar to that reported for branchedPEI-based DNA complexes, which have been used for intravenousadministration in Balb/c mice [26], indicating that serine-derivedsurfactant-based formulations possess dimensions suitable foruse in vivo. However, other routes for in vivo administration ofDNA complexes that are not constrained by their size have beenapplied [7,18,20]. Therefore, the potential of large DNA complexessuch as those based on (12Ser)2N5 for in vivo application shouldnot be excluded.

Another relevant feature presented by the complexes containingthe ester-derived surfactant is their negative Zeta potential at allcharge ratios tested (except at the highest charge ratio, in the pres-ence of helper lipids). Complexes containing the C12 amine-derivedsurfactant share this feature in most conditions. Such negativelycharged complexes should escape from the unspecific electrostaticinteraction with serum proteins, a drawback associated with theuse of highly positive charged particles for gene delivery in vivo.Although the transfection mediated by formulations containingthe amine and the amide derivatives was inhibited in the presenceof serum, the ester-containing complexes were able to transfectHeLa cells under these conditions, with no significant efficiencyreduction with respect to that obtained in the absence of serum.

Noteworthy, the high transfection efficiency of DNA complexesformulated with the ester derivative was observed at low surfac-tant/DNA charge ratios (1/1–2/1). This is particularly relevant fora potential application in vivo, since a decrease in surfactant doseensures a decrease in toxicity, thus reducing the safety concernsregarding the fate of the complex debris into the organism and inthe environment.

5. Conclusion

The most efficient complexes were those prepared with C12surfactants and helper lipids, reaching 50% of transfected HeLacells without causing cytotoxicity. These complexes have the par-ticularity of being most efficient at low surfactant/DNA chargeratios, which is advantageous in terms of cell, organism and envi-ronmental toxicity. Some of the most efficient complexes werefound to have sizes compatible with intravenous administration,as well as negative surface charge, which increases the likelihoodof reaching the desired target cells, and were shown to mediatetransfection even in the presence of serum. The most efficientand least cytotoxic complexes were produced from the biodegrad-able ester derivative, which supports the concept that cleavablechemical bonds would facilitate the release of DNA inside the cells.

Acknowledgments

The authors wish to acknowledge Prof. Graça Rasteiro, Depart-ment of Chemical Engineering of the Faculty of Science and Tech-nology, University of Coimbra, for the opportunity to use the Zetasizer device.

This work was funded by Portuguese Foundation for Scienceand Technology and FEDER/COMPETE through the followinggrants: PTDC/DTP-FTO/0265/2012 and PEst-C/SAU/LA0001/2013-14 (CNC, Univ. Coimbra); and PTDC/QUI-QUI/115212/2009 andPest/C-QUI/UI0081/2013 (CIQ, Univ. Porto). A.M.C., C.M.M. andS.G.S. are recipients of fellowships from the Portuguese Foundationfor Science and Technology (SFRH/BD/63288/2009, SFRH/BD/79077/2011 and SFRH/BD/61193/2009, respectively).

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