Acta Biomaterialia 10 (2014) 1412–1422

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Acta Biomaterialia

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Cyclen-based lipidic oligomers as potential gene delivery vehicles

1742-7061/$ - see front matter � 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.actbio.2013.12.010

⇑ Corresponding authors. Fax: +86 28 85415886 (X.-Q. Yu).E-mail addresses: ji_zhang@163.com (J. Zhang), xqyu@scu.edu.cn (X.-Q. Yu).

Wen-Jing Yi a, Qin-Fang Zhang a, Ji Zhang a,⇑, Qiang Liu a, Laifeng Ren b, Qian-Ming Chen c, Liandi Guo b,Xiao-Qi Yu a,⇑a Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu 610064, People’s Republic of Chinab Laboratory of Genome Stability, Development and Stem Cell Institute, The West China Second Hospital, Sichuan University, Chengdu 610041, People’s Republic of Chinac State Key Laboratory of Oral Diseases, Sichuan University, Chengdu 610041, People’s Republic of China

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

Article history:Received 7 June 2013Received in revised form 25 November 2013Accepted 9 December 2013Available online 14 December 2013

Keywords:Gene deliveryCyclenLipopolymerNon-viral vectorStructure–activity relationship

A series of cyclen-based linear oligomers bearing hydrophobic long chains (lipopolymers Cy-LC, where Cyand LC represent cyclen-based linear backbone and hydrophobic long chain substituents, respectively)were designed and synthesized. The effects of type and degree of substitution (DS) of hydrophobic longchains on the transfection efficiency were systematically studied. The nitrogen atoms with relativelystrong basicity on the cyclen ensure their good DNA binding ability, which was confirmed by gel retar-dation and ethidium bromide exclusion assays. Lipopolyplexes could be formed as nanoparticles withsuitable sizes and zeta potentials for gene transfection. In vitro gene delivery experiments revealed thatthe linoleic acid (LIN) substituted material Cy-LIN has better transfection efficiency than 25 kDa poly-ethylenimine in the absence or in the presence of serum. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide and hemolysis assays showed low cytotoxicity and good biocompatibility of thelipopolyplexes. Fluorescent labeled DNA was used to study the cellular uptake and intracellular distribu-tion of transfected DNA. Flow cytometry results suggested that a long chain is necessary for efficient cel-lular uptake, and images from confocal laser scanning microscopy showed that after 4 h transfection,most of the fluorescent labeled DNA accumulated in the perinuclear region, which was required for effi-cient gene expression. Moreover, it was also found that the DS of the hydrophobic moiety can adjust thebalance between DNA binding ability and dissociation of polyplexes, significantly affecting the transfec-tion efficiency.

� 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Safe and efficient delivery of genetic material remains the mostchallenging aspect of human gene therapy [1]. Although viral vec-tors are considered to have high gene transfer efficiency, theirapplications have been restricted by security issues such as immu-nogenicity, insertional mutagenesis, oncogenic effects and toxicity[2]. Non-viral gene carriers have been receiving increasing atten-tion over the past decades because of their low cost, flexibility inchemical design and safety [3–5]. Consequently, numerous non-viral gene delivery vectors have been developed and reported in re-cent years. These non-viral systems can be divided into liposomes(lipoplexes), polycationic polymers (polyplexes) and organic orinorganic nanoparticles (nanoplexes) [6,7]. However, it should benoted that although non-viral gene delivery vectors such as cat-ionic lipids and polymers have shown potential to overcome thesafety problems, low transfection efficiency (TE) limits their usein clinical trials [8].

The key steps involved in non-viral gene delivery include elec-trostatic complexation and condensation of DNA molecules intocompact particles, uptake of complexes by the target cells andendosome escape and dissociation of the complexes to releaseDNA to nucleus, which is necessary for expression of the deliveredgenes [9–11]. Strategies to improve the efficiency and biocompat-ibility of cationic reagents for gene delivery typically involve graft-ing functional ligands such as peptides [12], lipids [13,14], sugars[15] or a combination [2] to improve the stability, targeting, uptakeand sub-cellular trafficking capabilities of the vectors [16]. In thisregard, hydrophobic modifications of cationic regents could facili-tate DNA adsorption onto cellular surface by membrane fusion, andas a result, improve the endocytosis and TE [17]. Neamnark et al.reported that substitution of 2 kDa polyethylenimine (PEI) withaliphatic lipids including caprylic, myristic, palmitic, stearic, oleicand linoleic acids led to a TE comparable to 25 kDa PEI [18]. Withthe palmitic acid modification on poly-L-lysine), the productmaterial gave the TE that was equivalent to adenoviral carrier[19]. Le Gall et al. reported that cationic lipophosphoramide withdiunsaturated lipid chains could transfer the target gene into thelung with high efficiency in the in vivo gene delivery [20].

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Moreover, hydrophobic modifications of cationic reagents includ-ing chitosan [21], poly[2-(dimethylamino)ethyl methacrylate][22], spermine [23], etc., showed an approximate increase of TE,which is likely due to the balanced protection and release of DNAas well as to their function as membrane-anchoring moieties.Moreover, the alkyl chain length and degree of substitution (DS)may also affect some other properties such as cytotoxicity, biocom-patibility and in vivo circulation time [24]. A delicate balance be-tween hydrophilic and hydrophobic components is crucial for thedesign of new cationic carriers [25]. Optimal chain length and DSshould be selected by considering the overall effects of hydropho-bic modifications on the gene therapy [26]. However, there are fewreports of systematic investigation of the structure–activity rela-tionship of hydrophobic modified non-viral gene delivery vectors.

Our group previously reported a series of cyclen-based cationicpolymers showing comparable transfection efficiency to 25 kDaPEI with relatively lower cytotoxicity [27–29]. It is of great impor-tance to further investigate the structure–activity relationship ofthis type of cationic polymer. Herein we studied the effects ofthe structures and DS of lipidic moiety on the cyclen-based linearoligomer. Results show that the long chain modified complex couldtransfer target gene into several cell lines with higher TE and lowercytotoxicity compared to 25 kDa PEI, and the increased TE mightbe attributed to the enhanced cellular uptake and dissociation ofthe polyplexes inside the cell. We considered that these studieswould be particularly important to better understand the relation-ships between the lipid structure itself (length, amount, saturatedvs. unsaturated or polyunsaturated) and the physicochemicalproperties of the corresponding polyplexes, as well as their trans-fection activities.

2. Materials and methods

2.1. Materials and general measurements

Anhydrous ethanol, dichloromethane and epichlorohydrin weredried and purified under nitrogen by using standard methods andwere distilled immediately before use. 1,7-Bis (tert-butyloxycar-bonyl)-1,4,7,10-tetraazacyclodocane was prepared according tothe literature [30]. High molecular weight PEI (branched, averagemolecular weight 25 kDa: 25 kDa PEI), and MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchasedfrom Sigma–Aldrich (St Louis, MO, USA). The plasmids used inthe study were pGL-3 (Promega, Madison, WI, USA) coding forluciferase DNA and pEGFP-N1 (Clontech, Palo Alto, CA, USA) codingfor EGFP DNA. Cy™5 was obtained from Molecular Probe (Mirus,Madison, WI, USA). Dulbecco’s modified Eagle’s medium (DMEM),1640 medium and fetal bovine serum (FBS) were purchased fromInvitrogen Corp. MicroBCA protein assay kit was obtained fromPierce (Rockford, IL, USA). Luciferase assay kit was purchased fromPromega (Madison, WI, USA). Endotoxin free plasmid purificationkit was purchased from TIANGEN (Beijing, China). 293T humanembryonic kidney cell lines, A549 lung cancer cell lines andU2OS human osteosarcoma cancer cells were purchased from theShanghai Institute of Biochemistry and Cell Biology, Chinese Acad-emy of Sciences. All other reagents used in the synthesis, if notspecified, were obtained from Sigma–Aldrich and used withoutfurther purification.

Characterization and structural confirmation of intermediatesand products were performed by proton nuclear magnetic reso-nance (1H NMR) (Varian INOVA-400 spectrometer) and electro-spray ionization time-of-flight (ESI-TOF, Waters Q-TOF Premier)mass spectrometry. The molecular weights of the oligomers weredetermined by a Waters 717 plus autosampler (Waters Corp.,Milford, MA, USA) using tetrahydrofuran (THF) as eluent at a flow

rate of 1.0 ml min�1. Two Waters Styragel columns (HR3 and HR4)were used in series, and the detector (Waters 2414 refractive indexdetector) and columns were maintained at 45 �C throughout theruns. Linear polystyrene standards were used for calibration.100 ll of each sample prepared at 2 mg ml�1 was injected, andeach sample was given 40 min to elute off of the column. Fluores-cence spectra were measured by a Horiba Jobin Yvon Fluoromax-4spectrofluorometer.

2.2. Synthesis of lipid-substituted compound and polymers

2.2.1. Synthesis of lipid-diethanolamine compound (1)Aliphatic acid (caprylic, lauric, palmitic, stearic, oleic and lino-

leic acid, 0.04 mol) was dissolved in anhydrous dichloromethane(CH2Cl2, 50 ml). The solution was stirred at 0 �C for 10 min fol-lowed by the addition of isobutyl chloroformate (5.46 g,0.04 mol) and N-methylmorpholine (4.05 g, 0.04 mol). After addi-tion of diethanolamine (5.05 g, 0.048 mol), the solution was stirredat 0 �C for 30 min and at 25 �C for 6 h. The solution was washedwith sat. NaHCO3 (2 � 50 ml), and brine (2 � 50 ml). The organiclayer was dried over MgSO4, and concentrated under reduced pres-sure. The residue was purified by silica gel column chromatogra-phy to give compound 1, whose detailed analytical data arelisted in the Supplementary data.

2.2.2. Synthesis of epoxy-lipid-diethanolamine cross-linkers (2)Compounds 2 were prepared according to the literature [31]. In

a typical procedure, a mixture of epichlorhydrin (9.34 ml,0.12 mol), lipid-diethanolamine 1 (0.02 mol), sodium hydroxidepellets (4.8 g, 0.12 mol), water (1 ml, 0.056 mol) and tetrabutylam-monium chloride (0.3224 g, 0.001 mol) was vigorously stirred for2 h at 40 �C. The solid produced in the reaction was filtered offand washed with dichloromethane. The combined organic layerwas dried with anhydrous magnesium sulfate. The solvent and ex-cess epichlorhydrin were distilled off to give oily products. The res-idue was purified by silica gel column chromatography to give theproduct 2, whose detailed analytical data are listed in the Supple-mentary data.

2.2.3. Synthesis of hydrophobically modified polymer (Cy-LC)1,7-Bis(tert-butyloxycarbonyl)-1,4,7,10-tetraazacyclodocane

(223.5 mg, 0.6 mmol) was dissolved in 0.5 ml of C2H5OH, thencompound 2 (0.6 mmol) was added to the solution. Under the pro-tection of N2, the reaction mixture was stirred at 80 �C for 80 h. Thereaction mixture was stirred overnight after adding 50 ml satu-rated MeOH–HCl solution at room temperature and purifying bydialysis (MWCO 3.5 kDa) against pure water for 3 days. The reten-tate was lyophilized to give the product. Using 1H NMR (400 MHz,D2O), the integrals of the characteristic proton peaks of the substi-tuted fatty acids (d � 0.8 ppm, –CH3 and d � 1.3 ppm, –CH2) and ofpolymer backbone (d � 2.8–4.0 ppm) were obtained.

2.2.4. Synthesis of hydrophobically modified polymer with different DS(Cy-LIN0.5�3)

Cyclen-based linear oligomer (LCPA) (0.036 mmol) was pre-pared according to the literature [28], and was dissolved in CH2Cl2.The solution was stirred at 0 �C for 10 min followed by the additionof various amounts of linoleic acid (fatty acid:LCPA ratios = 0.5, 1, 2and 3), HOBt (N-hydroxybenzotriazole, 0.036 mmol) and EDCI(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,0.036 mmol). After addition of DIEA (N,N-diisopropylethylamine,0.036 mmol), the solution was stirred at 25 �C for 6 h. The solventwas removed under reduced pressure and added to 30 ml satu-rated MeOH–HCl solution at room temperature and purified bydialysis (molecular weight cut-off (MWCO) 1000 kDa) against purewater for 3 days. The retentate was lyophilized to give the product

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Cy-LIN0.5�3. Using 1H NMR (400 MHz, D2O), the integrals of thecharacteristic proton peaks of the substituted fatty acids(d � 0.86 ppm, –CH3; d � 1.27 ppm, –CH2 and d � 5.28 ppm,–CH@CH) and of polymer backbone (d � 2.5–4.1 ppm) wereobtained.

2.3. Gel retardation assay

Plasmid pEGFP-N1 (BD Biosciences Clontech) and pGL3-control(Promega) were amplified in Escherichia coli JM109 and extractedwith Endo-Free Plasmid Maxi Kit (E.Z.N.A.™, Omega). Polymers/DNA complexes were freshly prepared at various w/w ratios. 5 llcomplex solution containing 0.2 lg plasmid pGL3-control wasincubated for 30 min at room temperature and run on 1% (w/v)agarose gel at 80 V for 40 min. Gel was stained with ethidium bro-mide (EB) and the mobility of pDNA bands were analyzed using aMolecular Imager ChemiDoc XRS + (BIO-RAD, USA).

2.4. EB displacement assay

The ability of polymers to condense DNA was studied using EBexclusion assays [14]. Fluorescence spectra were measured atroom temperature in air by a Horiba Jobin Yvon Fluoromax-4 spec-trofluorometer and corrected for the system response. EB (2.5 ll,1 mg ml�1) was put into a quartz cuvette containing 2.5 ml of10 mM Hepes solution (pH = 7.4). After shaking, the fluorescenceintensity of EB was measured. Then CT DNA (10 ll, 1 mg ml�1)was added to the solution and mixed symmetrically, and the mea-sured fluorescence intensity is the result of the interaction be-tween DNA and EB. Subsequently, the solutions of polymer(1 mg ml�1, 2 ll for each addition) were added to the above solu-tion for further measurement. All the samples were excited at520 nm and the emission was measured at 600 nm.

2.5. Measurement of particle size and zeta potential

The complexes of polymers/DNA at various w/w ratios rangingfrom 2 to 16 were prepared as described above. 1 lg pGL3-controland the corresponding amount of polymers were respectively dis-solved in 100 ll PBS buffer (pH = 7.4), and mixed. After 30 minincubation at room temperature, polymers/DNA complexes werediluted with 900 ll H2O, and the zeta potentials and the hydrody-namic diameters were measured using a Malvern Zetasizer NanoZS (Malvern Instruments Ltd, UK) at 25 �C.

2.6. Transmission electron microscopy (TEM)

TEM images were obtained on a JEM-100CX (JEOL) transmissionelectron microscope at an acceleration voltage of 100 kV. The TEMsamples were prepared by dipping a copper grid with Formvar filminto the freshly prepared nanoparticles solution (1.0 lg ml�1). Afew minutes after the deposition, the aqueous solution was blottedaway with a strip of filter paper and then the samples were driedfor 2 min at room temperature. The samples were stained withphosphotungstic acid (ATP) aqueous solution and dried in air.

2.7. Cytotoxicity of the complexes evaluation

Cytotoxicity of polymer/DNA and PEI/DNA complexes wereevaluated by MTT assay. 1.5 � 104 cells per well 293T, U2Os andA549 cells were respectively seeded in 96-well plates. After 24 hculture, the lipopolyplexes (vector-DNA complexes) or free vectorswere added to cells at different chosen w/w ratios and concentra-tions. For complexes, the final pGL-3 concentration is 0.2 lg perwell in a total volume of 100 ll for another 24 h. After that, themedium was replaced again with 200 ll of fresh medium, and20 ll of sterile filtered MTT (5 mg ml�1) stock solution in PBS

was added to each well. After 4 h, unreacted dye was removedby aspiration. The formazan crystals were dissolved in 150 ll di-methyl sulfoxide per well and measured spectrophotometricallyin an ELISA plate reader (model 550, BioRad) at a wavelengthof 570 nm. The cell survival was expressed as follows: cellviability = (ODtreated/ODcontrol) � 100%.

2.8. Gene transfection efficiency assay in vitro

2.8.1. Gene delivery studies: expression of the Luc geneA549, U2Os and 293T cells were seeded at a density of 7 � 104

cells per well in a 24-well plate in DMEM (U2Os and 293T cells) orRPMI-1640 medium (A549 cells) containing 10% FBS and grown toreach 70–80% confluence prior to transfection. For transfection inthe absence of serum, the medium of each well was exchangedfor fresh serum-free medium. For transfection in the presence ofserum, the medium of each well was not changed at this time. Sub-sequently, the cells were treated with polyplexes (containing 1 lgof pGL3-control or pEGFP-N1) at different (w/w) weight ratios andPEI/DNA at weight ratio of 1.4 (N/P = 10) for 4 h at 37 �C. The med-ium was then completely refreshed with the completed culturemedia. After an additional 24 h incubation, the medium was re-moved and the cells were washed with 500 ll 1� PBS (pH 7.4)twice, lysed with 100 ll 1� lysis reporter buffer (Promega) andcentrifuged at 12,000g for 3 min at 4 �C. Luciferase activity in20 ll supernatant was evaluated with a luciferase assay system(Promega). The gene transfection efficiency of each sample wasrepresented by firefly luciferase expression and calculated as rela-tive light units per milligram of total protein (RLU/mg protein).Protein concentrations in cell lysates were determined using BCAProtein Assay Kit (Pierce) with bovine serum albumin (BSA) asstandard.

2.8.2. Gene delivery studies: expression of the EGFP geneEnhanced green fluorescent protein expression studies were

carried out as mentioned above for the Luc gene expression. 24 hafter transfection, cells were observed with an inverted fluores-cence microscope (Nikon Eclipse TE 2000E) equipped with a coldNikon camera. Digital image recording and image analysis wereperformed with the NIS Elements Advanced Research (version2.31) software.

GFP expressions were quantitatively measured using flowcytometry. The cell suspensions were evaluated using Aria 2 flowcytometry and 10,000 cells were evaluated in each experiment.Data acquisition and analysis were performed using Aria 2 flowcytometry (Becton, Dickinson and Company). GFP fluorescencefrom the expression of the plasmid DNA was measured in theFL1 channel using the 488 nm blue laser. Untransfected cells wereused to set the background.

2.9. Cellular uptake of plasmid DNA

The cellular uptake of the polymer/fluorescein labeled-DNAcomplexes was analyzed by flow cytometry. The Label IT Cy5Labeling Kit was used to label pDNA with Cy5 according to themanufacturer’s protocol. Briefly, A549 cells were seeded onto 12-well plates (1.6 � 105 cells per well) and allowed to attach andgrow for 24 h. For transfection in the absence of serum, the med-ium was exchanged with serum-free medium. As for transfectionin the presence of serum, the medium was exchanged with ser-um-containing medium. Cells were incubated with Cy5 labeledDNA nanoparticles (2 lg DNA per well, optimal N/P ratio of eachsample) in media for 4 h at 37 �C. Subsequently, the cells werewashed with 1� PBS and harvested with 0.25% trypsin/ethylenedi-aminetetraacetic acid and resuspended in 1� PBS. Meanfluorescence intensity was analyzed using FACSCalibur flow

W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422 1415

cytometer (Becton Dickinson and Company). Cy5-labeled plasmidDNA uptake was measured in the FL4 channel using the red diodelaser (633 nm). Data from 10,000 events were gated using forwardand side scatter parameters to exclude cell debris. The flow cytom-eter was calibrated for each run to obtain a background level of�1% for control samples (i.e., untreated cells).

2.10. Confocal laser scanning microscopy (CLSM) analysis

A549 cells were seeded at a density of 6 � 104 cells per wellonto the 24-well plate with a sterile cover glass (8 mm � 8 mm)in each well and incubated for 24 h. For transfection in the absenceof serum, the medium was exchange with serum-free medium.Complexes of polymers and Cy5-labeled pGL3 at a given concen-tration were added to each well. After 4 h, the cells on the coverglass were washed three times with PBS buffer and fixed with 4%paraformaldehyde (dissolved with PBS buffer) for 10 min; nuclearstaining was done with 40,6-diamidino-2-phenylindole (DAPI).The CLSM observation was performed using an Olympus FV1000– IX81 (Leica, Germany) with a 40� objective at excitation wave-lengths of 405 nm for DAPI (blue), 633 nm for Cy5 (red),respectively.

2.11. Hemolytic activity test

The hemolytic activity was measured based on the reportedmethod [32]. A total of 200 ll of diluted rabbit blood was addedto different polyplexes and the volume was adjusted to 1 ml withsterile PBS (pH = 7.4). 200 ll of rabbit blood mixed with 800 ll ofthe PBS served as the positive control, and 200 ll of rabbit bloodmixed with 800 ll of the PBS containing an excess of ammoniumchloride to cause complete hemolysis was used as the negativecontrol. After the vials were incubated at 37 �C for 90 min, the solu-tions were centrifuged at 2000 rpm for 10 min. A total of 200 ll ofthe supernatant was collected and seeded in each well of a 96-wellplate. The absorbance at a wavelength of 543 nm was recorded ona Synergy HT Multi-Mode Microplate Reader (BioTek, USA). Thepercentage hemolysis (PH%) (mean% ± SD, n = 3) was calculatedusing the following formula:

PH% = (As � Ap)/(An � Ap) � %, where As, An and Ap representthe absorbance of sample, negative and positive controls,respectively.

3. Results and discussion

3.1. Synthesis of the hydrophobic long chain modified oligomers Cy-LC

Scheme 1 summarizes the general synthetic methods for thehydrophobic-modified oligomers, which were named accordingto the abbreviations of the hydrophobic groups. Diol glycidylethers 2 with various long chains were first prepared to act asthe bridge molecule. Subsequent ring-opening polymerization tookplace between 1,7-diprotected cyclen and 2. The final deprotectionby HCl and dialysis gave the comb-like products Cy-LC. The dialysisyields of the oligomers were not greatly different, suggesting thatsimilar polymerization degrees were achieved for the six products.These new compounds were obtained as hydrochlorides and char-acterized by 1H NMR (400 MHz) using D2O as solvent. Since themolecular weights of Cy-LC could not be regularly measured bygel permeation chromatography (GPC), the indirect method wasused by employing Boc-substituted substrates, whose molecularweights were measured to be in the range of 2599–3603 (polydis-persity index: 1.1–1.2, Table S.1 of Supplementary data) by GPCusing THF as mobile phase, indicating an average of 4–5 repeating

units in one molecule. All of the target oligomers were found to bewell soluble in pure water.

3.2. Interaction with plasmid DNA

The ability of polymers to bind DNA to form polyplex is a pre-requisite for efficient DNA transfection. Gel electrophoresis hasbeen widely used to reveal the interaction between different poly-mers and DNA. As reported in previous work, hydrophobic moie-ties could facilitate a cooperative binding of polymer with DNA[33]. To identify the appropriate vector/DNA weight ratio requiredfor efficient condensation, a gel mobility assay was carried out. Asshown in Fig. 1A, full retardation of plasmid DNA was observedfrom the w/w ratio of 4. For compound Cy-LIN, the ratio for fullretardation was even lower. These results show that all the oligo-mers can effectively bind DNA at a relatively low dosage. On theother hand, the binding abilities of these vectors were further eval-uated through EB exclusion assay. The fluorescence intensity of EBis significantly increased by nucleic acid intercalation. When a cat-ionic agent binds to the plasmid, some of the intercalated EB issubstituted, resulting in measurable reduction of the fluorescenceintensity. Fig. 1B shows that the addition of Cy-LC to DNA pre-treated with EB caused considerable decrease of fluorescenceintensity, which was almost completely quenched at the w/w ratioof 2. Generally, the oligomer with the shorter aliphatic chainshowed the better fluorescent quenching ability. This might bedue to its weaker ability to screen the positive charge on the cyclenmoiety which was necessary for DNA condensation.

Having the proper size and shape of polymer/DNA complexes isessential for efficient gene delivery. The effective diameters of thevarious alkyl-modified polyplexes were measured by dynamiclight scattering (DLS). As shown in Fig. 2A, polyplex particles wereformed with the sizes in the range of 100–250 nm at different w/wratios. For oligomers with longer hydrophobic chain (Cy-STE, Cy-OLE and Cy-LIN), the 18-C length substituent might facilitate theformation of micelle-like structure, which would condense DNAmore compactly. Consequently, the average sizes of the polyplexesformed from these three vectors were distinctly smaller (<100 nm)than those from other ones with shorter hydrophobic chains, espe-cially at higher w/w ratios. As reported in the literature [2], theparticles with mean diameters between 70 and 90 nm are most lia-ble to the endocytosis. We also examined the possible changes ofparticle sizes of Cy-LIN/DNA with various w/w ratios in the pres-ence of 10% serum. Results suggest that the particle sizes of poly-plexes were �300 nm, and seldom altered with the increase ofw/w ratio, indicating that no apparent aggregation occurred(Fig. S.1, Supplementary data). Furthermore, the morphology ofthe Cy-LIN/DNA polyplex was observed at w/w ratios of 4 by trans-mission electron microscopy (TEM), and the image is shown inFig. 2B. The polyplex was well dispersed as individual nanoparti-cles in the range of 80–90 nm with a regular spherical shape. Fromthese results we considered that Cy-STE, Cy-OLE and Cy-LIN maycondense DNA to form nanoparticles with proper sizes for genedelivery. To our knowledge [34], the discrepancy of particle sizemeasured by DLS (135 ± 1 nm) and TEM (80–90 nm) could beattributed to the fact that DLS determines the hydrodynamic diam-eter of micelles in water, whereas TEM reveals the morphology ofthe micelles in the dehydrated state.

The zeta potentials of the polyplexes were measured at variousweight ratios ranging from 2 to 12 via DLS, and Fig. 2A shows theresults. At the weight ratio of 2, zeta potentials were found to benegative except that from Cy-CAP. This could also be attributedto its lower positive charge screening ability caused by the shorterhydrophobic chain. The zeta potentials rose along with the in-crease in weight ratio, and turned to be positive from the weightratio of 4. For the lipopolyplexes with saturated hydrophobic

Scheme 1. Strategy of the preparation of cyclen-based oligomers functionalized with various hydrophobic chains.

Fig. 1. (A) Agarose gel electrophoresis of varied alkyl-modified oligomers complexed with DNA to form polyplexes at different weight ratios. The first lane is DNA control.(B) Fluorescent quenching assay of EB/DNA by the addition of Cy-LC.

Fig. 2. (A) Mean diameter (columns) and zeta potential (dots) of Cy-LC/DNA complexes obtained at various weight ratios by DLS. (B) TEM image of Cy-LIN/DNA complexes ata weight ratio of 4.

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chains, a longer chain led to larger zeta potential at weight ratiosgreater than 2. On the other hand, polyplexes with unsaturatedhydrophobic chains (Cy-OLE and Cy-LIN) showed lower zetapotentials than the saturated ones, which might be due to thepositive charge screening by the electron-rich double bond.Generally, the positively charged complexes can interact with thenegatively charged cellular membranes, leading to efficient intra-cellular uptake of the complexes into the cells.

3.3. In vitro transfection by lipopolymers

The in vitro gene transfection efficiencies of the lipopolyplexeswere first studied in A549 cells using pGL3 plasmid as a luciferasereporter gene. Fig. 3A exhibits the TE at different weight ratios inserum-free medium, and 25 kDa PEI was used as control. Interest-ingly, the results demonstrate that the TE of the lipopolyplexes in-creased along with the increase of hydrophobic chain length. The

W.-J. Yi et al. / Acta Biomaterialia 10 (2014) 1412–1422 1417

unsaturated long chain seemed to play an important role in thetransfection process, and polyplexes from unsaturated chain mod-ified Cy-OLE and Cy-LIN gave considerable higher TE: for example,Cy-LIN gave �1.5 times higher TE than PEI at the weight ratioof 4. We speculate that the lower zeta potentials caused by theelectron-rich double bonds in Cy-LIN might benefit the dissocia-tion of the polyplexes, leading to better release of DNA in the cyto-sol. It was also reported that the linoleic acid substituents may helpto increase membrane-traversing motility, leading to increased up-take or additional intracellular events (e.g. endosomal escape ornuclear leakage) and subsequent better gene expression [18,35].In addition, since Cy-LIN has the largest molecular weight amongthe oligomers, this might also contribute to its higher TE. Eventhough these lipopolymers were prepared under identical reactionconditions, unfortunately it was difficult to obtain the target poly-mers with very similar molecular weights. Oligomer Cy-LIN wasused as a model compound in the subsequent studies. In general,the interaction between negatively charged serum proteins andthe cationic polyplexes would inhibit efficient gene delivery. Toestimate the in vivo transfection potential, we studied its genedelivery ability in the presence of serum. As shown in Fig. 3B,although the TE of Cy-LIN/DNA polyplexes was lower than that ob-tained in the absence of serum, it also exhibited higher TE than PEIat a weight ratio of 4, indicating its further applications as an effi-cient non-viral vector. Moreover, the TE of Cy-LIN in 293T andU2Os cell lines were also quantified by the same assay. Fig. 3Cshows that related to PEI, the TEs were greatly reduced in these celllines. These results suggest that Cy-LIN may have superior trans-fection ability toward lung cancer A549 cells. It is probably becausedifferent cell types possess varied transfection pathway and cell

Fig. 3. (A) Luciferase gene expression transfected by Cy-LC/DNA complexes at different wrepresent mean ± SD (n = 3). (B) Luciferase gene expression transfected by Cy-LIN/DNA apresence of serum in A549 cells. (C) Transfection in 293T and U2Os cells.

apoptosis features, and the biomedical nature of the gene deliveryvectors result in intrinsic physicochemical properties of complexes[36]. Compared to PEI, Cy-LIN has lower cytotoxicity in 293T cellsbut higher cytotoxicity in U2Os cells (Fig. S.2, Supplementary data).

Green fluorescent protein (GFP) assay was processed to directlyvisualize the infected cells by using pEGFP-N1 as reporter gene.The weight ratios were used according to the optimal results ofthe luciferase assay, and the images are shown in Fig. 4. Similarto the results in the luciferase assays, oligomer Cy-LIN gave thestrongest green fluorescence among the six polymers (A–F), andthe density of expressed GFP was also higher than that involvingPEI (F and G). Serum seems to have no negative effect on the trans-fection mediated by Cy-LIN (H); however, PEI-participated trans-fection was inhibited by serum (I). These results also confirmedthe higher biocompatibility of the lipopolyplexes compared toPEI, which has high positive charges. In 293T and U2Os cell lines,similar to the results of luciferase assay (Fig. 3C), Cy-LIN also gavelower GFP expression than PEI in the absence of serum (Fig. S.3,Supplementary data).

3.4. Cellular uptake and intracellular distribution

The hydrophobic interaction is known to play a key role in thebinding of amphiphilic compounds to biological membranes [24].It was considered that the hydrophobic modifications on cationicpolymers would facilitate the cellular uptake by membrane fusion.To further study the transfection mechanism involving the lipo-polymers, the fluorescence-activated cell sorting (FACS) techniquewas applied after incubation of A549 cells with the lipopolyplexescontaining Cy5-labelled DNA in the absence of serum. The

eight ratios in comparison with 25 kDa PEI (w/w = 1.4, N/P = 10) in A549 cells. Datat different weight ratios in comparison with 25 kDa PEI (w/w = 1.4, N/P = 10) in the

Fig. 4. Fluorescence microscope image of pEGFP-transfected A549 cells: (A–F) Cy-LC at a weight ratio of 4 in the absence of serum; (G) 25 kDa PEI (w/w = 1.4, N/P = 10) in theabsence of serum; (H–I) Cy-LIN (w/w = 4) and 25 kDa PEI (w/w = 1.4, N/P = 10) in the presence of serum.

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incubation was processed at the optimized weight ratio of 4 for 4 h.Fig. 5 shows the percentage of Cy5-positive cells and the meanfluorescence intensity of Cy5. It was found that hydrophobic longchain with 12 or more carbon atoms was necessary for efficientcellular uptake. Lipopolymers with 12 or 16 carbon atoms(Cy-LAU and Cy-PAL) gave similar positive cell percentage to PEI(87–90%), while the percentage was largely increased by using18-C contained vectors (up to 97.4%, especially for Cy-LIN), sug-gesting that aliphatic long chain (saturated or unsaturated) maybenefit membrane fusion and subsequent cellular uptake of thepolyplexes [36]. Saturated stearoyl substituted Cy-STE and unsatu-rated linoleoyl substituted Cy-LIN gave almost the same percent-age of Cy5-positive cells, and the fluorescence intensities weresimilar. However, Cy-LIN gave much higher TE than Cy-STE(Fig. 3A), indicating that the unsaturated part in the lipopolymermight have other unique properties which benefit the transfectionsteps other than cellular uptake.

Fig. 5. Cellular uptake (columns, percentage of Cy5-positive cells) and meanfluorescence intensity (dots) of lipopolyplexes (w/w = 4) in A549 cells quantified byflow cytometry analysis. 25 kDa PEI (w/w = 1.4, N/P = 10) was used as control. Thevalues were the means ± SD for three independent transfection repeats.

Subsequently, the intracellular distribution of Cy5-labeled DNAmediated by these lipopolymers was tailed by CLSM, and the nucleiwere stained with DAPI (blue). As shown in Fig. 6, considerableamounts of DNA (red) were delivered to the perinuclear region ofA549 cells following 4 h of incubation with Cy-LC/DNA polyplexes.As indicated in the flow cytometry assay (Fig. 5), a larger amount oflabeled DNA transferred by C18-long chain contained vectors wasfound compared to that mediated by shorter chain contained ones.It is worth mentioning that more Cy5-labeled DNA localized withinthe nuclei could be found in Cy-LIN-mediated transfection. For thetransfection by other lipopolymers such as Cy-LAU and Cy-STE,more Cy5-labeled DNA was found in the cytosol, but not nuclei.Since stronger nucleus association ability is correlated with bettertransgene expression, these results confirmed that linoleic acidmodified polymer Cy-LIN mediates superior intracellular DNAdelivery to the others including PEI 25 K [35,36]. After release ofpolyplexes into cytosol, Cy-LIN-mediated nuclear importationmight be achieved through hydrophobic interaction with nuclearmembrane.

3.5. Biocompatibility

Cytotoxicity is a crucial factor in assessing biosafety for biomed-ical application of cationic polymers [37]. The viability of A549cells by the treatment of polyplexes prepared at various weight ra-tios was studied by using branched 25 kDa PEI as control. As shownin Fig. 7, the cell viabilities for almost all lipopolyplexes were morethan 80% below the weight ratio of 8 after 24 h incubation, com-paring to �30% cell viability with the use of PEI under w/w of 6.We considered that the presence of hydroxyl and ether groups inthe backbone of the lipopolymers might reduce the net positivecharge and the cytotoxicity even at higher weight ratios. No dis-tinct relationship between the hydrophobic chain structures andcytotoxicity was found, and Cy-OLE seemed to be the most bio-compatible one among the six vectors. In addition, MTT assayswere also carried out to examine the cytotoxicity of free lipopoly-mers under various concentrations, and similar trends were found(Fig. S.4, Supplementary data).

Fig. 6. CLSM images of A549 cells transfected with Cy5-labeled DNA by Cy-LC at a weight ratio of 4 (PEI control at w/w of 1.4). For each row, left: cell nuclei stained by DAPI(blue); middle: Cy5-labeled pDNA (red); right: merged image.

Fig. 7. Cytotoxicity of Cy-LC/DNA polyplexes at different weight ratios toward A549cells. Data represent mean ± SD (n = 3).

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One of the large limitations on the in vivo application of cationicpolymers in gene delivery is the instability of polyplexes in blooddue to their nonspecific interactions with blood components. Thus,the hemocompatibility of the lipopolymers was evaluated by mea-suring their hemolytic activity [32]. Hemolysis of the erythrocytesis caused by the destabilization of their membrane, resulting in therelease of hemoglobin. It is generally assumed that the charge den-sity and the balance between hydrophobic and hydrophilic groupsof a cationic polymer are the factors regulating its hemolytic activ-ity. The relative hemolysis percentages compared to ammoniumchloride were determined with the lipopolymers concentration of8 lg ml�1. As shown in Fig. 8A, no evident hemolysis caused by

the lipopolymers and their corresponding polyplexes was observed(similar to PEI), indicating that these bioconjugates led to littleerythrocyte membrane disturbance. Fig. 8B shows the visual obser-vation of hemolysis caused by the lipopolyplexes and PEI in phos-phate buffer at pH 7.4. These results demonstrate that thesehydrophobic modified polycationic materials are highly blood-compatible for in vivo applications.

3.6. Linoleic acid substituted linear cyclen-based polyamine and theirgene delivery ability

Previous results revealed that the introduction of linoleoyl sub-stituent (LIN) may distinctly increase the TE of the cyclen-basedcationic polymer. To further investigate the TE and other proper-ties of the polymer with various degrees of LIN substitution, sev-eral LIN-modified linear cyclen-based cationic materials wereprepared [28]. As shown in Scheme 2, modification of LCPA withdifferent equivalent of LIN gave the product with DS of long chains.Since each LCPA monomer has three amine groups ready for acyl-ation, we prepared Cy-LIN0.5, Cy-LIN1, Cy-LIN2, Cy-LIN3 by usingthe mole ratio (LIN/LCPA monomer) of 0.5, 1, 2, 3, respectively.The DS were calculated according to 1H NMR spectra of each prod-uct as 0.54, 1.02, 1.68 and 2.61 for Cy-LIN0.5–Cy-LIN3, respectively,which were similar to the LIN equivalents used in the reactions.Polymers with higher DS exhibit lower hydrophilicity and there-fore lower water solubility.

The DNA condensation ability of Cy-LINx was also estimated bygel retardation assay and EB displacement assay. Fig. 9A clearlyshows that the DNA retardation ability decreased along with theincrease in DS, and Cy-LIN0.5 gave the strongest DNA binding abil-ity, which could completely retard DNA at a weight ratio of 2. Thisresult was reasonable since Cy-LIN0.5 has the lowest DS and highest

Fig. 8. (A) Hemolysis of the lipopolymers alone and their polyplexes with DNA. (B) Visual observation of hemolysis caused by Cy-LC/DNA in phosphate buffer at pH 7.4.Rabbit blood containing 8 lg ml�1 of lipopolymers or its complexes with DNA at a weight ratio of 4 and excess of ammonium chloride was separately incubated at 37 �C for90 min and then centrifuged at 2000 rpm for 10 min.

Scheme 2. Preparation of linoleic acid substituted LCPA with different DS.

Fig. 9. (A) Agarose gel electrophoresis of LA-modified polymers complexed with DNA to form polyplexes at different weight ratios. (B) Fluorescent quenching assay of EB/DNA by addition of Cy-LINx.

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positive charge density. The steric hindrance caused by the longchain might be another reason for the decreased DNA binding abil-ity of the polymer with higher DS. Similar regularity was found inthe EB displacement assay (Fig. 9B). Cy-LIN0.5 could almost quenchthe fluorescence intensity at weight ratio of 1, while Cy-LIN3

needed a weight ratio of 4 to achieve the same result.The properties including particle size, shape and zeta potential

of the polyplexes formed from Cy-LINx and DNA were also exam-ined by DLS and TEM. Results revealed that with the increase ofDS, the particle size also increased and the zeta potential was re-duced. This might be attributed to the greater amount of LIN moi-ety, which increases the molecular volume and screens the positivecharge of the cationic polymer. MTT and hemolysis assays showedthat these polyplexes have similar biocompatibility compared toCy-LC (Figs. S.5 and S.6, Supplementary data).

The p-EGFP transfection efficiencies of Cy-LINx and PEI towardA549 cells were quantified by flow cytometry analysis. Cy-LIN0.5–Cy-LIN3 gave the best TE at a weight ratio of 12, 8, 8 and 12, respec-tively (data not shown for the transfection under other w/w ratios).As shown in Fig. 10A, the DS of hydrophobic moiety plays animportant role in the gene delivery, and the highest percentageof fluorescence-positive cells was obtained by using Cy-LIN2, whichgave the amount of transfected cells �1.6 times more than PEI.Further, a larger difference was found for the mean fluorescenceintensity caused by Cy-LIN2 and PEI (�24,592 vs. �7245), indicat-ing that higher expression per cell could be achieved by the lipo-polyplex. The fluorescence microscope images gave morevisualized pEGFP transfection results, which were consistent withthe flow cytometry analysis (Fig. S.7, Supplementary data).Whether or not the transfection was carried out with serum, the

Fig. 10. (A) Transfection efficiency of Cy-LINx toward A549 cells by flow cytometry. TE was indicated by the percentage of pEGFP-expressing cells (columns) and meanfluorescence intensity of expressed GFP (dots). (B) Cellular uptake (columns, percentage of Cy5-positive cells) and mean fluorescence intensity (dots) quantified by flowcytometry. The values were the means ± SD for three independent transfection repeats. (C) CLSM images of A549 cells transfected with Cy5-labeled DNA by Cy-LIN2. Left: cellnuclei stained by DAPI (blue); middle: Cy5-labeled pDNA (red); right: merged image.

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density of transfected cells by Cy-LIN2/DNA was distinctly higherthan that by PEI/DNA polyplex. It was considered that the DSmay affect the balance between DNA binding ability and dissocia-tion of polyplexes. Lipopolymers with lower DS may have toostrong DNA affinity, which makes it difficult to release DNA inthe cytosol. On the other hand, higher DS may result in weakDNA binding and low lipopolyplex stability. The cellular uptakepercentages of Cy-LINx/DNA polyplexes were also quantified byflow cytometry using Cy5-labelled DNA. As shown in Fig. 10B,except Cy-LIN0.5 (87.4%), other three lipopolymers with higher DSgave higher cellular uptake (>95%) than PEI, indicating that thehydrophobic chain has a significant positive effect on theendocytosis by membrane fusion. CLSM was also used to studythe intracellular distribution of Cy5-labeled DNA transfected byCy-LINx, and Fig. 10C shows the typical results involving Cy-LIN2.It was shown that after 4 h transfection, most of the fluorescentlabeled DNA was accumulated in the perinuclear region, whichwas necessary for efficient gene expression.

4. Conclusions

In this report, LCPA was modified with hydrophobic long chainsthrough two different methods. One is the polymerization betweendiBoc-protected cyclen and long chain contained bridge, and theother is direct acylation of LCPA. The former method helps us tostudy the effect of different hydrophobic chains, and the lattermethod gives a way to investigate the effect of the DS on genedelivery. Compared to LCPA, the hydrophobic modified materialsgave much higher TE, which was also higher than 25 kDa PEI.The structure–activity relationship of the lipopolymers wasstudied, and the long chain group (especially linoleic acid substitu-ent) was found to be crucial for efficient cellular uptake andnuclear orientation. Furthermore, it was also found that the DS of

hydrophobic moiety plays an important role in the gene delivery,and it may affect the balance between DNA binding ability anddissociation of polyplexes. Good TE together with excellent bio-compatibility mean that this type of lipopolymer is promising asa non-viral gene delivery vector. Subsequent studies will focuson further development of the lipopolymers, such as the introduc-tion of biodegradable or cell-targeting moieties.

Acknowledgements

This work was financially supported by the National Programon Key Basic Research Project of China (973 Program,2012CB720603 and 2013CB328900), the National Science Founda-tion of China (No. 21232005) and State Key Lab of Oral Diseases(Sichuan University) (SKLODSCUKF2012-02). J. Z. thanks theProgram for New Century Excellent Talents in University (NCET-11-0354). We also thank Analytical & Testing Center of SichuanUniversity for structural analysis of the compounds.

Appendix A. Figures with essential colour discrimination

Certain figures in this article, particularly Figs. 1–10 are difficultto interpret in black and white. The full colour images can be foundin the on-line version, at http://dx.doi.org/10.1016/j.actbio.2013.12.010.

Appendix B. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.actbio.2013.12.010.

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