Nuclear localisation and pDNA condensation in non-viral gene delivery

THE JOURNAL OF GENE MEDICINE R E S E A R C H A R T I C L EJ Gene Med 2007; 9: 265–274.Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1015

Nuclear localisation and pDNA condensation innon-viral gene delivery

Elizabeth Collins1,2

James C. Birchall1

Julie L. Williams2

Mark Gumbleton1*

1Welsh School of Pharmacy, CardiffUniversity, Cardiff, UK2Pfizer Global Research &Development, Sandwich, UK

*Correspondence to:Mark Gumbleton, Welsh School ofPharmacy, Redwood Building, KingEdward VII Avenue, Cardiff CF103XF, UK. E-mail:[email protected]

Received: 13 June 2006Revised: 20 December 2006Accepted: 12 January 2007

Abstract

Background Non-viral gene delivery vectors are multi-component systemsreflecting various functionalities required for effective cell transfection,including DNA condensation, promotion of cell membrane interactionsand provision for subcellular targeting through endosomal escape and/ornuclear delivery. Elements mediating these functions will clearly displayinter-dependency. In this study we sought to explore the relationship withinnon-viral vectors of condensation and nuclear localisation.

Methods Binary, tertiary and quaternary vectors were prepared withcombinations of pDNA, DOTAP lipid, the polycation peptide protamineand either SV40 nuclear localisation sequence peptide (‘SV40 NLS’) or aone amino acid substituted mutant of SV40 NLS (‘mutant sequence’). Theefficiency of pDNA condensation was determined by gel electrophoresis andquantitative fluorescence spectroscopy. Transfection efficiency was examinedin mammalian cells in vitro using standard methods, by electroporation tobypass the plasma membrane barrier and in cells arrested in G0/G1 cell cyclephase to examine the effect of cell division and nuclear membrane disruption.

Results Small NLS peptide sequences, despite possessing a significantproportion of basic amino acids, display minimal pDNA-condensing abilitywhen compared to larger polycations such as protamine. In standardin vitro cell adherent transfection studies the predominant elements affordingenhanced gene expression were effective pDNA condensation and lipidenhancement of cell membrane interactions. These features conversely hinderefficient gene expression in cells that have undergone electroporation. Thebenefit of SV40 NLS was only apparent when used in non-dividing cellpopulations.

Conclusions Whilst effective levels of non-viral-mediated gene expressiongenerally rely on efficient condensation of pDNA and enhanced interactionswith cellular membranes, non-covalently associated NLS within a multi-component non-viral gene vector appears to contribute benefit in sustaininggene expression in non-dividing cells. Copyright 2007 John Wiley & Sons,Ltd.

Keywords nuclear localisation; pDNA condensation; non-viral; gene delivery;electroporation; cell cycle

Introduction

The challenge of delivering efficient non-viral gene therapy lies inthe production of a synthetic virus-like complex that has the abil-ity to effectively transfer the gene in an active form to both

Copyright 2007 John Wiley & Sons, Ltd.

266 E. Collins et al.

dividing and non-dividing cells. Numerous polymers andpeptides have been included in non-viral gene vectorsto facilitate gene transfer and most contain a plasmidDNA (pDNA)-condensing moiety to reduce the size, andincrease the protection of, the nucleotide cargo. Suchmaterials include, among others, synthetic copolymers[1–4], branched or linear homopolymers [5,6] andpolymers coupled to receptor targeting ligands [7,8].In each case the cationic vector self-assembles withanionic pDNA through electrostatic interactions to formcondensed particles.

In order to facilitate efficient gene expression the deliv-ered pDNA must initially circumnavigate the variousbarriers to cellular and nuclear entry. The aforementionedpDNA-condensing elements appear to play an intimaterole in negotiating various cellular barriers. Nucleocyto-plasmic transport occurs across the nuclear membranethrough nuclear pore complexes (NPC). Under conditionsof active transport the NPC can accommodate particlesup to 25 nm in diameter [9,10]. This affords access tothe nucleus of critical regulatory macromolecules that areunable to passively diffuse through the NPC but exploitactive transport via the presence, within their proteinchain, of a nuclear localisation sequence (NLS). In genetherapy studies NLS peptides have been investigated asfacilitators of nuclear transport with the aim of enhanc-ing transgene expression. The amino acid sequence ofthe first NLS to be established by point mutation, SV40large T antigen, contains a high percentage of basic aminoacids; practically all NLSs identified since have been foundto contain a high proportion of cationic amino acids.Such peptides therefore can encompass a dual role, firstlyas potential nuclear signalling moieties, and secondlythrough the positively charged amino acids which serveto neutralise and condense the pDNA cargo [11–13].However, it is acknowledged that condensation of pDNAalone, even in the presence of an NLS peptide, is generallynot sufficient to mediate efficient transgene expression.Additional vector components including cationic lipids[12,13] and fusogenic lipids [14] have been investigatedas components to enhance efficient cellular delivery. Asan example, tertiary formulations comprising pDNA, apolycationic condensing peptide and cationic lipid, i.e. alipid : polycation : pDNA (LPD) vector, continue to receivesignificant attention [15–18], with protamine sulphatebeing one of the most commonly used condensing poly-cations [19–26].

Some previous reports have stated that NLS peptidesconfer no significant increase on the transgene expressionof non-viral DNA carrier systems [27,28] and suggestthat any increased transfection efficiency is attributed toincreased compaction/protection of pDNA rather that anactive localisation to the nucleus [29]. A recent paperby Akita and co-workers [30] evaluated the role of achimeric vector comprising SV40 NLS conjugated to µ(mu) peptide in condensing pDNA and facilitating nucleartransport in vitro. In their conclusion the authors assertthat it is the polycation µ (mu) sequence acting as acondensing element, and not the SV40 NLS, that is

primarily responsible for efficient nuclear transfer of intactpDNA. They relate their studies to previous work with thispeptide that reached similar conclusions [29,31]. In thiscurrent work we aim to further investigate the interactionbetween condensing moieties, nuclear transport peptidesand pDNA and their effect on transfection efficiency inmammalian cell lines. Specifically, we sought to clarifywhether SV40 large T antigen (SV40 NLS), a mutant non-signalling sequence (mutant sequence), and protaminesulphate elicit any nuclear localisation properties whencomplexed to pDNA, and explore the relationship betweenpDNA condensation and nuclear localisation. To addresssome of the specific issues raised previously [30], weemployed an alternative strategy using a non-covalentlyattached NLS in the presence or absence of additionalcondensing moieties. In our studies electroporation isused to bypass plasma membrane transfer of the pDNAcondensate. The nuclear membrane is investigated as abarrier to efficient transfection by halting cells in G0/G1

phase of cell cycle and so inhibiting mitosis and nuclearmembrane disruption.

Materials and methods

Materials

The following compounds were used as received: agaroseLE (Promega, Southampton, UK), ethidium bromidesolution (Pharmacia Biotech, St Albans, UK), sodiumdodecyl sulphate (SDS) (Pharmacia Biotech); Tris base,Luria Bertani (LB) agar, LB broth, HEPES buffer (H-7523) (all Sigma-Aldrich Chemicals, Poole, UK). Cellculture plastics were obtained from Corning-Costar (HighWycombe, UK). Dulbecco’s modified Eagle’s medium(DMEM 25 mM HEPES), foetal bovine serum (FBS),penicillin-streptomycin solution and trypsin-EDTA solu-tion 1X were obtained from Life Technologies (Paisley,UK). All other reagents were of analytical grade and pur-chased from Fisher Scientific UK (Loughborough, UK).

Plasmids

The 4.7 kb pEGFP-N1 plasmid construct containing thegreen fluorescent protein (GFP) reporter gene waspropagated and purified as detailed previously [24] usingkanamycin to ensure selective growth of transformedbacteria.

Peptides and lipids

1,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP)was purchased from Avanti Polar Lipids (Alabama, USA).Liposomes were prepared by reconstitution of a dried lipidfilm and extrusion (The Extruder, Lipex BiomembranesInc., Vancouver, Canada) through 100 nm polycarbon-ate filters (Millipore, Watford, UK). Protamine sulphate

Copyright 2007 John Wiley & Sons, Ltd. J Gene Med 2007; 9: 265–274.DOI: 10.1002/jgm

Condensation and Localisation of Vectors 267

grade X from salmon sperm was obtained from SigmaChemicals (Poole, UK). SV40 large T antigen NLS (SV40NLS: PKKKRKV) and a one amino acid substituted mutantsequence peptide (mutant sequence: PKTKRKV) were cus-tom made by Peninsula Laboratories Europe Ltd. (StHelens, UK).

Condensation of pDNA by polyaminoacids and peptides

The degree of condensation afforded by cationic peptidesand polycations was investigated using both agarose gelretardation and a quantitative fluorometric assay. Bothmethods exploit the intercalation of ethidium bromide(EtBr) with pDNA to produce a fluorescent signal underUV light. Upon condensation of pDNA, EtBr cannot readilyaccess the base pairs and a reduction in fluorescent signalis observed.

Gel electrophoresis assaySamples were prepared by adding the appropriate amountof peptide (SV40 NLS, mutant sequence or protaminesulphate) to 2 µg of pDNA (pEGFP-N1) to a final volumeof 20 µL (ddH2O). The samples were incubated for 5 minprior to addition of 4 µL loading dye (0.25% bromophenolblue, 0.25% xylene cyanol FF, 30% glycerol in water).The peptide : pDNA complexes were analysed using a 1%agarose gel prepared in 0.5xTBE buffer. The gel was runat 150 V for 2 h, stained with 0.5 µg/ml EtBr solution andanalysed using a Gel Doc 1000 with Molecular Analyst

software (Bio-Rad Laboratories, Hercules, CA, USA).

Spectrofluorometric assayPlasmid DNA (pEGFP-N1; 15 µg) was incubated in a 3 mlcuvette with EtBr (3.5 µg) and the resultant fluorescence(Aminco Bowman spectrofluorometer utilising SLM-AB2software) taken as 100%. Peptide (SV40 NLS, mutantsequence or protamine sulphate) was incrementally addedto a final volume of 3 ml. After each addition of peptidethe sample was mixed by inversion, covered with foiland incubated at room temperature (RT) for 3 min priorto measuring fluorescence. Fluorescent readings werecorrected for dilution and expressed as a percentage ofthe signal attributed to pDNA alone. The wavelengthsfor excitation and emission were 516 nm and 598 nm,respectively, with corresponding slit width set at 4 nm. Allmeasurements were performed in 25 mM HEPES buffer(pH 7.4) and at a temperature of 25 ◦C.

Cell culture

The mammalian cell lines, A549 and COS-7 (EuropeanCollection of Animal Cell Cultures, Salisbury, UK),were cultured in 24-well format with culture mediacomprising DMEM, 10% FBS, and the antibioticspenicillin (100 IU/ml) and streptomycin (100 µg/ml).

Cells (75 200/well) were grown to 80% confluency at37 ◦C in a humid atmosphere at 95% air/5% CO2.

Cell transfection

Cells were surface-rinsed twice with phosphate-bufferedsaline (PBS) and exposed to gene vectors in DMEM(equivalent to 5 µg pDNA per well). Following incubationat 37 ◦C for 6 h the cells were surface-rinsed again withPBS and fed with 1 ml of culture media (DMEM/10%FBS/antibiotics). The cells were returned to the incubatorfor a further 42 h to allow intracellular expression of theplasmid to proceed.

Flow cytometric quantification of geneexpression

Following surface rinsing with PBS, transfected cellswere trypsinised with 0.3 ml trypsin-EDTA solution,and resuspended in 0.6 ml culture media (DMEM/10%FBS/antibiotics). The percentage of cells displaying GFP-associated fluorescence (FL1-H) was quantified by flowcytometry (FACScan; Becton Dickinson Immunocytome-try Systems, San Jose, CA, USA) with analysis by WinMDISoftware (Joseph Trotter, The Scripps Institute, La Jolla,CA, USA), as described previously [24].

Electroporation of the MCF-7 cell line

MCF-7 cells (gift from W. Jiang, Department of Surgery,School of Medicine, Cardiff University, UK) were seededin 75 cm2 flasks and grown in culture media (DMEM/10%FBS/antibiotics) and a 5% CO2 humidified atmosphere.The cells were removed from the flasks using trypsin-EDTA and suspended to a concentration of 5 millioncells/ml in DMEM/10% FBS. Complexes were preparedby adding the appropriate amount of cationic peptideto 30 µg pDNA to a total volume of 100 µl (ddH20).Then DMEM (700 µl) was added to each complexmixture. The cells were centrifuged at 700 rpm for10 min and re-suspended with the complex suspension.The cells/complex were transferred to an electroporationcuvette and electroporated at voltage 250 V and capacity1500 mF (Easyject T Plus, Equibio Ltd, Kent, UK). Twenty-four hours after electroporation 63% of MCF-7 cells wereviable (assessed using trypan blue) compared to 93% ofcells which had been trypsinised but not electroporated(data not shown). Immediately after electroporation thecells were transferred to warm culture media (5 ml)and incubated in 24-well format at 37 ◦C for 24 h.Subsequently, the cells were washed with PBS, removedfrom the wells with trypsin-EDTA, suspended in culturemedia and subjected to GFP quantification by flowcytometry.



Cell cycle arrest and quantification ofGFP expression

MCF-7 cells and the non-tumor BEAS-2B human bronchialepithelial cell line were seeded in 24-well format ata density of 40 000 cells/cm2 and grown for 24 h ina 5% CO2 humidified atmosphere in culture media(DMEM/10% FBS/antibiotics). Media was removed fromthe cells and fresh media returned to the control cellswhilst FBS-free media was returned to the cells to behalted in G0/G1 phase. The cells were left to cultureunder these conditions for a further 24 and 48 h. Forflow cytometry, cells were washed and removed fromwells using trypsin-EDTA (neutralised with culture media)prior to preparation of cells for cell cycle analysis usingthe CycleTEST reagent kit (Becton Dickinson) with post-acquisition flow cytometric data analysed by FACScan andModfit software (Verity Software House Inc., Topsham,ME, USA) and Cylchred cell software.

For transfection, following 24 h of serum depletionthe media was removed, each well was surface-rinsedtwice with pre-warmed PBS (pH7.4) and re-fed withDMEM alone. Complexes were prepared by adding theappropriate amount of cationic peptide and lipid to 30 µgpDNA to a total volume of 300 ml (ddH2O). To eachcomplex DMEM (5.7 ml) was added. DMEM was removedfrom each well and replaced by 1 ml of the complexsuspension (5 µg pDNA/well) and the cells incubated at37 ◦C, 5% humidified CO2 for 6 h. After 6 h the DMEM,containing the treatments, was removed from the cells andreplaced with either serum-plus or serum-deficient mediaas appropriate. The cells were analysed for expression ofGFP 18 h post-transfection.

Results

The effect upon pDNA condensation of increasing the ratioof cationic peptide to anionic pDNA is shown initially inFigure 1; lanes 1 and 17 show migration of naked pDNAalone. Increasing the charge ratio (from 0.5 : 1 to 6 : 1+/−) of the SV40 NLS peptide (lanes 2–6) and mutantsequence peptide (lanes 7–11) to the anionic pDNAappears to have a minimal effect upon the exclusion ofEtBr from binding to the DNA as evidenced by the intensityof the migrating bands, and thus indicating that thepDNA does not become significantly condensed by thesesequences. Compared with the mutant sequence (lanes7–11), pDNA complexation with SV40 NLS (lanes 2–6)leads to slightly more retardation of pDNA movementthrough the gel, presumably through shielding of thenegative charge on the phosphate groups of pDNA. Incontrast, even at a charge ratio of 0.5 : 1 (+/−), protaminesulphate significantly retards pDNA signal on the gel (lane12) with most of the signal retained within or close tothe loading well. When the protamine to pDNA chargeratio is increased to 2 : 1 (+/−) and above, the protaminecomplexes the pDNA to such an extent as to almost

SV40 NLS mutant sequence protamine

Figure 1. Extent of EtBr exclusion by cationic peptides – gel elec-trophoresis assay. SV40 NLS (amino acid sequence PKKKRKV),a single amino acid substituted mutant sequence peptide(PKTKRKV) or protamine sulphate was mixed with pDNA atincreasing charge (+/−) ratio, incubated for 5 min and analysedon a 1% agarose gel, stained with EtBr and visualised underUV light. Ratios are expressed as peptide : pDNA charge ratio(+/−). Lanes 1, 17: uncondensed pDNA control; lane 2: SV40NLS : pDNA 0.5 : 1; lane 3: SV40 NLS : pDNA 1 : 1; lane 4: SV40NLS : pDNA 2 : 1; lane 5: SV40 NLS : pDNA 4 : 1; lane 6: SV40NLS : pDNA 6 : 1; lanes 7–11: mutant sequence : pDNA chargeratios (+/−) as lanes 2–6; lanes 12–16: protamine : pDNAcharge ratios (+/−) as lanes 2–6. Reverse contrast imagedisplayed

completely exclude EtBr, as evidenced by the lack offluorescent signal (lanes 14–16).

The extent of pDNA condensation by the cationicpeptides was further investigated using a quantitativespectrofluorometric assay. The data shown in Figure 2is consistent with the gel electrophoresis observations inthat protamine achieves a greater than 90% reduction influorescence signal intensity (signal intensity proportionalto EtBr intercalation with DNA) at a protamine : pDNAcharge ratio as low as 2 : 1 (+/−). This degree ofcondensation is clearly not matched with the use ofSV40 NLS or the mutant sequence, with the SV40 NLSmediating approximately 40% reduction in signal at anequivalent (2 : 1) charge ratio, and this level of EtBrexclusion essentially remaining unchanged even whenincreasing SV40 NLS to provide a charge ratio (+/−) ashigh as 6 : 1. With the mutant sequence the reductionin fluorescence signal intensity did not exceed 20% at acharge ratio of 6 : 1 (+/−).

The effect of elements within a multi-componentgene delivery vector that mediate pDNA condensation,nuclear localisation and lipid-mediated enhancement ofmembrane interactions was examined by flow cytometry(Figure 3). In either cell line tested (A549 or COS-7) no significant difference (P < 0.05) was observedin gene expression efficiency between untreated cellsand those treated with ‘naked’ pDNA, or indeedthose treated with peptide : pDNA complexes (eitherprotamine, SV40 NLS or mutant sequences) preparedat a 2 : 1 (+/−) charge ratio. In each case, however, atertiary formulation comprising lipid : peptide : pDNA wastransfection-competent and superior (P < 0.05) to therespective non-lipid-containing vectors. Nevertheless, inthese lipid-containing formulations the presence of SV40NLS provided no advantage (P > 0.05) over the mutant



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Figure 2. Extent of EtBr exclusion by cationic peptides – spectro-fluorometric assay. EtBr (3 µg) was added to pDNA (15 µg)and the resultant fluorescence measured. The peptides(♦ = protamine sulphate, � = SV40 NLS, � = mutant sequence)were added incrementally at concentrations equivalent to (+/−)charge ratios ranging from 0.25 to 6. The fluorescence was mea-sured and expressed as a percentage of the initial fluorescenceattributed to EtBr intercalation of naked pDNA. Data representedas mean ± standard deviation (s.d.), n = 3

peptide sequence. Further, the tertiary formulationcontaining lipid : protamine : pDNA (LPD 0.7 : 2 : 1 (+/−)complex) exhibited higher (P < 0.05) gene expressionefficiency than the corresponding tertiary formulationscontaining either the SV40 NLS or mutant sequences.All the above tertiary formulations were more effective(P < 0.05) transfecting agents than a respective bi-component lipid : pDNA (0.7:1 +/−) vector.

To explore the influence of NLS per se on gene expres-sion, but nevertheless attempt to account for the relativelypoor condensation properties of SV40 NLS, a quater-nary complex comprising lipid : protamine : NLS : pDNAwas formulated. Protamine sulphate was added topDNA at charge ratios of either 2 : 1 or 1 : 1 (+/−;protamine : pDNA) prior to addition of the SV40 NLS(Figure 4B) or mutant sequence (Figure 4C) (thesesequences added at a fixed charge ratio of 2:1 +/−of peptide : pDNA). A final addition to the above was ofDOTAP cationic lipid (at a fixed charge ratio of 0.7:1+/− of lipid : pDNA) to provide quaternary formulationsof lipid : protamine : NLS : pDNA. Tertiary vectors used ascontrol complexes (Figure 4A) comprised two formula-tions of DOTAP : protamine : pDNA with varying contentof protamine and providing formulations of charge (+/−)ratios of 0.7 : 2 : 1 (control 1) and 0.7 : 1 : 1 (control 2).

We found that reducing the protamine : pDNA chargeratio within the tertiary lipopolyplex from 2 : 1 (control1) to 1 : 1 (control 2) resulted in a decrease (P < 0.05) ingene expression efficiency from 60% to 49% (Figure 4A).Quaternary vectors containing SV40 NLS (Figure 4B)or mutant sequence (Figure 4C) also showed maximumtransfection efficiency when protamine was incorporatedat the higher amount to give a protamine : pDNAcharge ratio of 2:1 +/−. However, the levels of geneexpression attained by these quaternary vectors werelower (P < 0.05) than the corresponding tertiary control

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Figure 3. Reporter gene expression from bi-component andtri-component vectors in A549 and COS-7 cell lines. Cells weregrown to 75% confluency and exposed to lipopolyplex complexescontaining 5 µg pDNA (except blank control) for a period of 6 h.The cells were washed, fed with culture medium and culturedfor a further 42 h. The percentage of cells showing greenfluorescent protein (GFP)-associated fluorescence (FL1-H) wasquantified by flow cytometry. A marked region was establishedcontaining 1% of the population of the untreated control cellsand the percentage of cells moving into this marked regioncalculated. The ratios indicated are charge (+/−) ratios. Datarepresented as mean ± s.d., n = 12. Statistical analysis byone-way analysis of variance (ANOVA) and Duncan’s multiplerange test. Significance level P < 0.05. Lipid = DOTAP; mutant= mutant sequence; NLS = SV40 NLS sequence; LPD =DOTAP : protamine : pDNA

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Figure 4. Transfection efficiency of tertiary and quaternarycomponent lipopolyplexes in the A549 cell line. Cells weregrown to 75% confluency and exposed to lipopolyplex complexescontaining 5 µg pDNA for a period of 6 h. The cells were thenre-fed and cultured for a further 42 h prior to analysis for thepresence of GFP. The ratios indicated are charge (+/−) ratios.Data represented as mean ± s.d., n = 12. Statistical analysis byone-way ANOVA and Duncan’s multiple range test. Significancelevel P < 0.05. Lipid = DOTAP; mutant = mutant sequence; NLS= SV40 NLS sequence

formulations. Again, at the higher protamine content theSV40 NLS provided no greater transfection benefit overthe mutant sequence.

Electroporation of MCF-7 cells was employed tobypass the plasma membrane barrier and determinethe gene expression of complexes delivered directly to



the cell cytoplasm. We found no significant (P > 0.05)difference in reporter gene (GFP) expression efficiencybetween pDNA that was electroporated alone, i.e. ‘naked’pDNA, and electroporated pDNA that was pre-complexedwith either NLS or mutant sequence (Figure 5A). Allof these treatments however demonstrated significantly(P < 0.05) enhanced gene expression compared toelectroporation of pDNA pre-complexed with protaminealone, or pDNA pre-complexed with protamine andDOTAP lipid. This suggests that the greater levelof condensation that provided a positive element totransfection when an intact plasma membrane was inplace serves as a hindrance when the plasma membranebarrier is bypassed. This is further exemplified inFigure 5B which compares the absolute percentage ofcells expressing GFP when transfected under standardcell culture conditions and following electroporation.Less than 1% of cells express GFP when naked pDNAis transfected under standard conditions but followingelectroporation this increases (P < 0.05) to ∼20%. Incontrast transfection with the LPD tertiary vector (controlformulation 1) resulted in ∼26% of cells expressingGFP under standard conditions which reduces (P < 0.05)to 11% following electroporation, indicating that whenthe cell membrane no longer serves as a barrier thecomplexation of pDNA itself can ultimately hindertransfection efficiency.

To determine the effect of cell division, and hencenuclear membrane integrity, upon transfection efficiencyMCF-7 cells were arrested in G0/G1 phase by serumdeprivation. Standard flow cytometric analysis was usedto confirm that MCF-7 cells were predominantly haltedin G0/G1 (Figure 6). Following 24 h serum deprivation,approximately 90% of cells were in G0/G1 phase(Figure 6D) vs. 44% in G0/G1 for cells grown inthe presence of serum; however, after 48 h of serumdeprivation (Figure 6E), increased cell death was evidentrepresented by a more diffuse population of cells to theright of the G2/M region. Accordingly, subsequent cellcycle studies were restricted to cells deprived for serumfor only 24 h. Similarly, BEAS-2B cells when cultured inserum-free conditions for 24 h showed a doubling in the% cells halted in G0/G1 phase.

Figure 7 shows that under standard cell transfec-tion conditions (when the MCF-7 cells are subconflu-ent and actively dividing) a tertiary vector comprisinglipid : protamine : pDNA (LPD) exhibits greater transfec-tion efficiency compared to tertiary complexes preparedfrom lipid, SV40 NLS (or mutant sequence) and pDNA.This is consistent with data shown in Figure 3. How-ever, a significant (P < 0.05) decrease in LPD-mediatedgene expression efficiency was observed when cells weretransfected predominantly in the G0/G1 phase (Figure 7,G0/G1), i.e. an effective decrease in transfection of 42%in non-dividing cells. A decrease (P < 0.05; greater than50%) in transfection in non-dividing cells was also evidentfor the lipid : peptide : pDNA complexes substituted withthe non-functional mutant sequence peptide. In contrast,

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Figure 5. Electroporation in MCF-7 cells. Data representedas mean ± s.d., n = 12. Statistical analysis by one-wayANOVA and Duncan’s multiple range test. Significance levelP < 0.05. (A) MCF-7 cells were grown to confluence ingrowth media, trypsinised, aliquoted into microcentrifuge tubesand centrifuged. Gene vectors were prepared as describedpreviously with 30 µg of pDNA. GFP expression was analysed24 h after electroporation. (B) MCF-7 cells were transfectedunder standard conditions, i.e. in conventional adherent cellculture, or by electroporation, and analysed 24 h later. mutant= mutant sequence; NLS = SV40 NLS sequence; LPD =DOTAP : protamine : pDNA

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Figure 6. Flow cytometry dotplot representation of MCF-7 cellcycle and the effects of serum deprivation. Plots A, B and C showthe cell cycle populations for cells maintained in full growthmedia. Plots D and E show the cell cycle populations whereserum has been removed from the feeding media for 24 and48 h, respectively. For flow cytometry, cells were trypsinised,treated with ribonuclease A, stained with Triton-100/EtBr andanalysed by FACScan using Modfit

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when comparing dividing to non-dividing cultures trans-fected with the lipid : SV40 NLS : pDNA complex then nodecrease (P > 0.05) in gene expression was observed, i.e.



an equivalent level of transfection was maintained evenwhen the cells were not dividing. Further, in the G0/G1

cultures a clear difference (P < 0.05) can be seen betweenthe vectors containing the SV40 NLS and the mutantpeptide sequences. Although cells arrested in interphase(G0/G1) may take up macromolecules and DNA throughendocytosis it is possible that this could be at a lower levelthan cellular internalisation seen in actively dividing cells[41], and thus the intracellular quantities of DNA may bereduced in non-dividing cells. As this will be consistent foreach experimental treatment, however, our data can beinterpreted as the SV40 NLS, even when non-covalentlyassociated within a multi-component formulation, provid-ing some functionality in cells not actively replicating butlacking any significant impact in those cells undergoingmitosis.

Figure 8 shows substantiation of the cell cycle arrestdata shown above but using the non-tumor cell line BEAS-2B. Specifically, Figure 8 shows the transfection efficien-cies of the various formulations in BEAS-2B cells underconditions of serum-containing and serum-free culturewhere in the latter the cells were predominantly haltedin G0/G1 phase. Consistent with data from Figure 7, thetertiary complex comprising lipid : NLS : pDNA performedsignificantly (P < 0.05) better than the comparative ter-tiary formulation containing the mutant peptide sequence(lipid : Mutant : pDNA) resulting in a 2-fold greater levelof transfection in the growth-arrested cells. It is alsoof note that under serum-free conditions it was thetertiary system of lipid : NLS : pDNA, but not the com-parative lipid : mutant : pDNA formulation, that was able

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Figure 7. Comparison of GFP expression when lipopolyplexvectors were used to transfect control MCF-7 cells and MCF-7cells halted in G0/G1 by serum deprivation. Lipopolyplexes wereprepared at a lipid : peptide : pDNA charge ratio of 0.7 : 2 : 1(+/−). Data represented as mean ± s.d., n = 6. Statisticalanalysis by one-way ANOVA and Duncan’s multiple rangetest. Significance level P < 0.05. Lipid = DOTAP; mutant= mutant sequence; NLS = SV40 NLS sequence; LPD =DOTAP : protamine : pDNA

to achieve similar degrees of gene transfection efficiencycompared to both quaternary systems containing pro-tamine (Figure 8). Further, similar to the findings forMCF-7 cells, it is the lipid : NLS : pDNA formulation that isable to minimise the effective decrease in gene transfec-tion when moving from serum-containing to serum-freeconditions where the predominant proportion of cellsare halted in G0/G1, i.e. the fall in gene transfectionefficiency is >70% (P < 0.05) for the LPD vector, 60%

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Figure 8. Comparison of GFP expression when lipopolyplex vectors were used to transfect control BEAS-2B cells (serum + ve) (blackcolumn infill) and BEAS-2B cells halted in G0/G1 by serum deprivation (lined column infill). Tertiary complexes were prepared ata lipid : peptide : pDNA charge ratio of 0.7 : 2 : 1 (+/−). Quaternary complexes were prepared at a lipid : protamine : peptide : pDNAcharge ratio of 0.7 : 2 : 2 : 1 (+/−). Data represented as mean ± s.d., n = 4. Statistical analysis by one-way ANOVA and Duncan’smultiple range test. Significance level P < 0.05 and shows significant falls in the serum-ve data to the serum + positive control data.Lipid = DOTAP; mutant = mutant sequence; NLS = SV40 NLS sequence; LPD = DOTAP : protamine : pDNA



(P < 0.05) for the lipid : mutant : pDNA formulation, but<35% (P > 0.05) for the lipid : NLS : pDNA formulation.Further, the gene transfer efficiency of quaternary vec-tors comprising either lipid : NLS : protamine : pDNA orlipid : mutant : protamine : pDNA under both conditionsof serum deprivation (where the cells are predominantlyin G0/G1) and serum presence indicated that the benefitof NLS over a non-functional mutant peptide is no longerevident (Figure 8), with decreases in the serum-ve cellsof >60% compared to the control serum + ve conditions.This is consistent with the equivalency of quaternary sys-tem gene transfer we report in Figure 4 for A549 cellsstudied under serum-containing conditions. These latterfindings comparing NLS and mutant peptide functionwithin quaternary systems bearing protamine may notbe that surprising as protamine provides a greater pDNAcondensation, a factor which may outweigh any benefitof NLS functionality even in growth-arrested cells.

Discussion

Reported studies in cell cultures using SV40 large Tantigen as a nuclear localisation signal (NLS) haveproduced equivocal conclusions regarding the ability ofthe NLS peptide to increase gene expression. In thisstudy our aim was to elucidate under what conditionsnon-covalently associated SV40 NLS, when part of a multi-component pDNA gene delivery complex, can function asa NLS in vitro, and relate the effect to that achievedthrough simple electrostatic condensation of pDNA.

A critical feature of an effective DNA delivery vectoris the packaging or condensation of the therapeuticgene to afford smaller size and importantly protectionagainst nuclease attack. The properties of SV40 NLSare not favourable from a pDNA-condensing perspectivewhen compared with polycationic protamine sulphate.In this study we have confirmed using an agarose gelexclusion and migration assay, and by a quantitativespectrofluorometric assay, that NLS peptides such as SV40do not significantly affect the access of EtBr to pDNA,i.e. do not bring about significant pDNA condensation;similar findings have been reported previously [29,32].Quantitative spectrofluorimetry showed that protamine(32 residue peptide comprising 21 basic amino acidsall of which are arginine) when mixed with pDNA at acharge (+/−) ratio of 2 : 1 produces a greater than 90%reduction in EtBr intercalation. In contrast an equivalentcharge ratio using SV40 NLS peptide (7 residue peptidecomprising 5 basic amino acids, 4 lysine and 1 arginine)and the mutant peptide (7 residue peptide comprising 4basic amino acids, 3 lysine and 1 arginine) mediated onlyapproximately 40% and 10% reductions, respectively.

Protamine is recognised as an effective condensingagent for DNA. In the body protamines are specific tothe male germ cell and replace, as DNA-condensingspecies, histones present in somatic cell nuclei. Insperm nuclei the basic amino acid to phosphate ratio

is close to unity with almost complete neutralisationof negative charge. Atomic force microscopy [33] hasshown DNA-protamine condensates to display toroidmorphology with vertical stacking of condensed DNA.X-ray diffraction studies have shown several differentarginine-containing peptides, including protamine, tostabilise the B-form of DNA [34,35] and with diffractionpatterns interpreted as the binding of protamine to themajor groove of DNA [34]. Structural studies examiningthe interaction of DNA-condensing proteins from thesperm of different species have shown that such proteins(including protamine) possess potential to adopt α-helicalconformation. Increasing peptide length and the presenceof multiple arginine residues appear to afford greaterpotential for α-helix formation, and for interaction withDNA [35]. The short 7 amino acid sequences representingthe SV40 NLS or mutant sequence while comprisinga high proportion of basic amino acids clearly differfrom protamine with respect to their short length andreduced potential for secondary structure. Such peptidesalso contain as their major basic residue lysine.

It is clear that elements within non-viral gene deliverysystems desirable for enhancing effective gene expressionwould include an efficient condensing agent, a compo-nent which enhances interaction with cell membranesand various targeting or release motifs enabling enhancedendosomal escape and/or nuclear delivery. Condensa-tion of DNA appears critical to successful transfectionacross an intact plasma membrane. In an attempt tocompensate for DNA condensation, or the lack thereof,and provide for an opportunity to assess NLS function-ality, we formulated a variety of tertiary and quaternarynon-viral delivery systems. In all cases incorporation of arelatively small quantity of lipid to the peptide : pDNAcombinations increased transfection efficiency signifi-cantly. Although in each case a tri-component formulationof lipid : peptide : pDNA was superior to the non-lipidpeptide : pDNA vectors, it was nevertheless the protamine-containing tertiary formulation and not the correspondingformulation containing SV40 NLS peptide sequence thatconsistently produced a greater reporter gene expres-sion under standard transfection conditions; the SV40NLS peptide : lipid formulation offering no greater effi-ciency in transfection than when using the non-functionalmutant peptide sequence. Given that each of the ter-tiary formulations represent the same overall positiveto negative charge, the greater reporter gene expressiondemonstrated with the protamine-containing formulationwas consistent with the apparent greater degree of con-densation (protection) offered by this particular moleculeover the SV40 NLS and mutant peptide sequences. Indeedfurther emphasising the important role of the degree ofpDNA condensation, significant decreases in gene expres-sion were apparent when reducing the protamine : pDNAcharge ratio within the tertiary lipopolyplexes from 2 : 1to 1 : 1.

Studies with quaternary complexes comprising lipid:protamine:pDNA, with addition of either NLS or mutantsequences, were undertaken to explore the influence



of NLS per se on gene expression while effectivecondensation was achieved through the incorporation ofprotamine into the formulation. Again quaternary vectorsshowed the better transfection efficiency when protaminewas incorporated at the higher (+/−) ratio. Thisfurther emphasises the central role of protamine in suchformulations. Additionally, regardless of the combinationof protamine and SV40 NLS or mutant sequenceutilised, lipid : protamine : pDNA at a charge ratio of0.7 : 2 : 1 (+/−) produced the highest gene expressionand emphasises that even with additional condensingcapacity through the supplement of protamine, the SV40NLS sequence did not afford increased transfection ability.

Electroporation of MCF-7 cells was used to bypass theplasma membrane barrier to deliver vectors directly tothe cell cytoplasm. Approximately 20% of cells expressedGFP when naked pDNA alone was electroporated com-pared with <1% when pDNA alone was incubated withcells under standard non-electroporated transfection con-ditions. Of note was the lack of any improved transfectionefficiency over naked pDNA when electroporating pDNAcomplexed to either SV40 NLS or mutant sequence pep-tide. Other reports have indicated minor increases in geneexpression of electroporated pDNA in the presence of NLS,albeit using a linear DNA-NLS conjugate [28]. Takentogether this is initially suggestive of electroporation-mediated disassociation of the non-covalently attachedDNA-NLS complexes used in our study, thus preventingany NLS functionality within the cell. The lack of function-ality with respect to NLS we observe is however consistentwith previous studies that have showed no enhancementof gene expression capability of pDNA in the presenceof NLS following inter-cytoplasmic microinjection [36].These electroporation studies were performed in divid-ing cells and our data obtained with cultures wherecells were predominantly halted in G0/G1 impacts uponthe interpretation of the NLS functionality. Interestingly,the more highly condensed protamine : pDNA and thelipid : protamine : pDNA complexes displayed even lowerexpression than naked pDNA (or SV40 NLS : pDNA com-plexes) in the electroporated cells. This is consistent withthe conclusions drawn by Akita et al. [30] undertakingcytoplasmic and nuclear microinjection studies of variouspDNA : peptide complexes, and reasoning that efficientcondensation of pDNA by peptide results in a failure inrelease of nucleic acid hindering the progress of transcrip-tion.

The majority of in vitro studies using non-viralgene transfer agents are performed using cells thatare still actively dividing and hence encompass acompromised nuclear membrane. Conversely, manytarget tissues within the body would predominantlycontain differentiated non-dividing cells. To determinethe effect of cell division, and hence nuclear membraneintegrity, upon the efficiency of our various complexes weundertook studies in MCF-7 cells arrested in the G0/G1 cellcycle phase by serum deprivation. Following 24 h serumdeprivation, approximately 90% of cells were in G0/G1

phase without any supplementary cell death evident.

In control cells undergoing mitosis the protamine-containing LPD complexes exhibited greater transfectionefficiency compared to corresponding complexes preparedfrom SV40 NLS or mutant sequence. However, in G0/G1-arrested cells a significant decrease in LPD-mediatedgene expression efficiency was observed, as indeedwas also noted for the tertiary vector containing themutant peptide sequence. Our results are in agreementwith other researchers [37–41] who also suggest thatconventional non-viral gene delivery systems, such as theLPD complex, are inefficient in transfecting non-dividingcells. In contrast, the lipid : SV40 NLS : pDNA complexwas uniquely able to display comparable transfectionefficiency in both cells undergoing mitosis and indeedthose arrested in G0/G1. This is interpreted as thefunctional NLS having benefit to overall gene deliveryin cells not actively replicating but in contrast lacksany significant impact in the cells undergoing mitosis.The MCF-7 cell cycle arrest data was substantiatedin the non-tumor cell BEAS-2B where, in growth-arrested cultures, the tertiary complex comprisinglipid : NLS : pDNA performed significantly better than thecomparative tertiary formulation containing the mutantpeptide sequence (lipid : mutant : pDNA).

Clearly, pDNA condensation has a major effecton the physicochemical properties of non-viral genetransfer vectors and their ability to be protectedagainst nucleases and to interact efficiently with thecell membrane. Electroporation studies suggested thatLPD non-viral vectors require initial interaction withthe cell membrane, or vesicles derived thereof, inorder to ultimately release the pDNA for transcription.Conversely, incompletely condensed vectors such as SV40NLS : pDNA showed efficient gene expression followingelectroporation suggesting that the cell membranerepresents the primary rate-limiting barrier for thesesystems. When cells were halted from progressingthrough mitotic events the functionality of the SV40large T antigen NLS appeared to be confirmed. Thesignificant reduction in gene expression efficiency of theLPD complex under these conditions suggests that thenuclear membrane represents the rate-limiting barrier toconventional condensed non-viral gene delivery vectors.

Acknowledgements

The authors acknowledge the financial support of Pfizer GlobalResearch & Development, Sandwich, UK, for the PhD studentshipto E.C.

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Nuclear localisation and pDNA condensation in non-viral gene delivery

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