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Title Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET

Author(s) Shaheen, Sharif M.; Akita, Hidetaka; Yamashita, Atsushi; Katoono, Ryo; Yui, Nobuhiko; Biju, Vasudevanpillai;Ishikawa, Mitsuru; Harashima, Hideyoshi

Citation Nucleic Acids Research, 39(7), e48https://doi.org/10.1093/nar/gkq1327

Issue Date 2011-04

Doc URL http://hdl.handle.net/2115/45389

Type article

File Information NAR39-7_e48.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

https://eprints.lib.hokudai.ac.jp/dspace/about.en.jsp

Quantitative analysis of condensation/decondensation status of pDNA in the nuclearsub-domains by QD-FRETSharif M. Shaheen1, Hidetaka Akita1, Atsushi Yamashita2, Ryo Katoono2,

Nobuhiko Yui2,*, Vasudevanpillai Biju3, Mitsuru Ishikawa3 and Hideyoshi Harashima1,*

1Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University,Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, 2School of Materials Science, Japan Advanced Institute ofScience and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 and 3Nanobioanalysis Team,Health Technology Research Center, National Institute of Advanced Industrial Science and Technology,2217-14 Hayashi-cho, Takamatsu, Kagawa 761-0395, Japan

Received April 23, 2010; Revised November 25, 2010; Accepted December 14, 2010

ABSTRACT

Recent studies indicate that controlling the nucleardecondensation and intra-nuclear localization ofplasmid DNA (pDNA) would result in an increasedtransfection efficiency. In the present study, weestablished a technology for imaging the nuclearcondensation/decondensation status of pDNA innuclear subdomains using fluorescence resonanceenergy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeledpolycations as acceptor. The FRET-occurringpDNA/polycation particle was encapsulated in anuclear delivery system; a tetra-lamellar multifunc-tional envelope-type nano device (T-MEND),designed to overcome the endosomal membraneand nuclear membrane via step-wise fusion.Nuclear subdomains (i.e. heterochromatin and eu-chromatin) were distinguished by Hoechst33342staining. Thereafter, Z-series of confocal imageswere captured by confocal laser scanning micros-copy. pDNA in condensation/decondensation statusin heterochromatin or euchromatin were quantifiedbased on the pixel area of the signals derived fromthe QD and rhodamine. The results obtainedindicate that modulation of the supra-molecularstructure of polyrotaxane (DMAE-ss-PRX), a con-denser that is cleaved in a reductive environment,conferred euchromatin-preferred decondensation.This represents the first demonstration of the

successful control of condensation/decondensationin specific nuclear sub-domain via the use of an arti-ficial DNA condenser.

INTRODUCTION

In post-genome era, gene therapy is one of the new-generation approaches for the cure of intractablediseases, which can complement the defective gene (1–3).Since viral gene carrier potentially has adverse effects suchas oncogenicity (4) and inflammation (5), efficientnon-viral carrier is highly desired. It is generally con-sidered that transfection activity is rate limited by avariety of intracellular process such as endosomal escapeand nuclear transfer (6). Recent quantitative comparisonof intracellular trafficking of plasmid DNA (pDNA) andadenovirus collectively revealed that post-nuclear deliveryprocesses (i.e. transcription and translation) are dominantrate-limiting processes for the 2- or 3-order of magnitudeless transfection efficiency in non-viral vectors (7–9).Further mechanism-based studies clarified that pDNAtransfected with lipoplex mainly exist as condensed formin the nucleus, which prevents it from the access to tran-scription factors (7). In contrast, adenovirus genomicDNA is efficiently released in the nucleus. Moreover,adenoviral genome DNA was specifically localized onthe euchromatin region (7), where transcription activityis relatively active (10–13).Herein, we established an imaging technology for

visualizing and quantifying nuclear decondensation inspecified nuclear sub-domains by means of fluorescence

*To whom correspondence should be addressed. Tel: +81 11 706 3919; Fax +81 11 706 4879; Email: [email protected] may also be addressed to Nobuhiko Yui. Tel: +81 761 51 1640; Fax +81 761 51 1645; Email: [email protected]

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

Published online 1 February 2011 Nucleic Acids Research, 2011, Vol. 39, No. 7 e48doi:10.1093/nar/gkq1327

� The Author(s) 2011. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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resonance energy transfer (FRET) between quantum dot(QDs)-labeled pDNA as a donor, and rhodamine-labeledpolycation as an acceptor.FRET is a promising technique for monitoring

decondensation processes in cells, that occurs betweendonor–acceptor dyes tagged on single DNA molecules(14), or between dyes separately tagged on pDNA and acarrier (15,16). Of note, an analysis of FRET in thenucleus indicated that pDNA transfected with linearpolyethylenimine or histidylated polylysine are present inthe nucleus in a condensed form (15). To explain geneexpression in terms of intranuclear disposition, quantifica-tion of the condensation/decondensation status in morespecified intranuclear regions is required. In the presentstudy, we established a technique for quantifying pDNAin a condensed or decondensed form in heterochromatinand euchromatin. In this system, we used QD as a donor,since it has been successfully used as a probe for theanalysis of intracellular trafficking of macromolecules,including DNA in the past (17,18). Although CdSe-QDhas some cytotoxicity due to the release of cadmium ionsunder UV-radiation (19), fluorescent CdSe/ZnSnanocrystals have been reported to be ideal for the usein long-term, non-toxic imaging of nuclear targeting inliving cells (20), since they show superior brightness andresistance to photobleaching, compared to organicfluorophores. These unique characteristics facilitate theidentification of the nuclear localization of pDNA andavoid the risks associated with loss of DNA-derivedsignals by repeated excitation when Z-stack images areobtained. It is also noteworthy that the emission spectraof QDs can be easily modulated by changing the size ofthe QD, thus permitting spectral overlap to be optimized(21). The quantification of intracellular and intra-nucleardecondensation by monitoring FRET QD-labeled pDNAand acceptor-labeled polycations has been reported previ-ously (22). In the present study, we advanced this ana-lysis by one-step further, characterizing the nuclearsub-domain to which the DNA was routed.For an evaluation of the nuclear dispositions in relation

to the transgene expression, efficient nuclear deliverysystem is prerequisite. For this purpose, a pDNA/polycation core was encapsulated in multilayered lipidenvelope-type nanoparticle was used, which we refer toas a tetra-lamellar multi-functional envelope-type nanodevice (T-MEND) (23). In this system, condensed DNAcores are coated with two nuclear membrane-fusogenicinner envelopes and two endosome-fusogenic outerenvelopes, both of which are designed to overcomethe endosomal membrane and nuclear membranes,respectively.Using the present method, we were able to demonstrate

the first successful control of a condensation/decondensation profile in the intra-nuclear sub-domainusing a synthetic polycation, polyrotaxane (PRX), whichpossessed a necklace-like structure composed ofdimethylaminoethyl-modified a-cyclodextrin; DMAE-a-CD) and a disulfide-introduced PEG chain (15). Torender it bio-cleavable, the end of the PEG chain wascapped via disulfide bonding (Figure 1a). The flexiblemovement of a-CDs along with the PEG thread facilitates

electrostatic interactions between pDNA and PRXmultivalently by avoiding spatial mismatching (24).Thus, the method is useful in investigating the mechanismof extensive gene expression, and in understanding therate-limiting processes to be overcome for furthergene expression from the point of view of intra-nucleardisposition.

MATERIALS AND METHODS

Materials

1.2-Dioleoyl sn-glycero-3-phosphatidylethanolamine(DOPE) was purchased from Avanti Polar lipids(Alabaster, AL, USA). Cardiolipin (CL) and phosphatidicacid (PA) were purchased form Sigma (St Louis, MO,USA). Protamine sulfate salmon mint was purchasedfrom Calbiochem (Darmstadt, Germany). Plasmid DNApCMV-luc encoding luciferase was prepared using anEndoFree Plasmid Mega Kit (Qiagen, Germany). Stearyloctaarginine (STR-R8) (25) and cholesteryl-GALA (26)was custom-synthesized by NeoMPS (NeoMPS Inc., SanDiego, CA, USA). HeLa cells were obtained from theRIKEN Cell Bank (Tsukuba, Japan) and Health ScienceResearch Resources Bank (Osaka, Japan), respectively. Inthis study, we used luciferase (GL3)-inserted plasmidDNA. To prepare the luciferase-encoding vector, aninsert fragment encoding luciferase (GL3) was obtainedby Hind III/Xba I digestion of the pGL3-basic vector(Promega, Madison, WI, USA), and ligated to theHindIII/Xba I digested site of pcDNA3.1 (Invitrogen,Carlsbad, CA, USA) as reported previously (8,27).QD-545 streptavidin was obtained from Invitrogen.

Preparation of polyplexes of pcDNA 3.1-luc andpolyrotaxanes

In order to prepare pDNA core particle with DMAE-ss-PRX, 100 ml of naked pDNA solution (0.1 mg/ml in10mM HEPES) was added to 100 ml of polycationsolution as demonstrated previously (28). The concentra-tion of the DMAE-ss-PRX (Cp) at charge ratios wascalculated using the following equation:

Charge ratio (� Þ ¼ fCp �NDMAE=MWpg=ðCD=MWDÞ,

where, NDMAE denote the number of DMAE (16 in16DMAE-ss-PRX and 46 in 46DMAE-ss-PRX) residuesin the polycation molecule. The charge ratio (±) denotesthe ratio of number of positive charges derived fromamino groups of DMAE-ss-PRX to that of negativecharges on phosphate groups of DNA. MWP and MWD

denote the molecular weight of the polycation(16DMAE-ss-PRX: 24600, 46DMAE-ss-PRX:29200) andone nucleotide (average: 308), respectively.

CD denotes the concentration of pDNA (0.1 mg/ml). Inthe present study, we prepared the condensed core atcharge ratio of 5. In this condition, a 2.4 ml aliquot ofpDNA (2.5 mg/ml) (0.1mg/ml final concentration) wasmixed with a 9 ml aliquot of 10mM of DMAE-unitsDMAE-ss-PRX (final concentration 1mM of DMAEunits). The diameter and zeta-potential of the T-MEND

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were determined using an electrophoretic light-scatteringspectrophotometer (Zetasizer; Malvern Instruments Ltd,Malvern, WR, UK). As a reference of gene transfection,pDNA was also condensed with protamine at charge ratioof 2.2 as described previously (27).

Preparation of T-MEND

Lipid films composed of DOPE/CL=5:5 (total lipidcontent: 0.55mmol) were hydrated with 1ml of 10mMHEPES buffer (pH 7.4) for 10min at room temperature.The hydrated lipid film was then sonicated using aprobe-type sonicator (BRANSON Digital Sonifier-250;

Danbury, CT, USA) with an amplitude of 25% for10min to form single unilamellar vesicles (SUVs1).Similarly, SUVs was prepared with DOPE withphosphatidic acid (PA) of 7:2 ratio (total lipid content:0.55mmol) and additional 1% mol of cholesterol-modified GALA peptide (SUVs2). Condensed DNA par-ticles prepared above were mixed with SUVs1 at a ratio of2:1 (v/v) to coat the condensed DNA particles with adouble-lipid envelope as described previously (29,30). Astearylated octaarginine (STR-R8) solution (20% mol oftotal lipid) was added to the suspension of double-layerednuclear membrane-fusogenic particles. Then, this suspen-sion of nuclear membrane-fusogenic particles was mixed

(a)

(b) (c)

Figure 1. Schematic diagram illustrating technologies for intracellular trafficking and programed decondensation. (a) Structure of DMAE-ss-PRXand rhodamine-labeled DMAE-ss-PRX. It has an orientation, which resembles to a necklace-like structure composed of dimethylaminoethyl-modified a-cyclodextrin; DMAE-a-CD and a disulfide-introduced PEG chain. Moreover, the end of the PEG chain was capped via disulfidebonding. The number of threaded a-CDs and the molecular weight of PEG in the polyrotaxanes were 18 and 4000, respectively. (b) The strategyto deliver pDNA to the nucleus via a series of membrane fusions using T-MEND. pDNA is condensed with a polycation and then coated with anucleus-fusogenic lipid membrane (inner) and an endosome-fusogenic lipid membrane (outer). The surface is modified with a high density ofoctaarginine (R8), which marks the liposomes for internalization into cells via macropinocytosis (40). During stepwise fusion, the pDNA/polycationcore is released from the lipid envelope, which is advantageous for efficient transcription. (c) Transfection activity of T-MENDs encapsulating pDNAcore particles formed with DMAE-ss-PRXs and protamine. HeLa cells were transfected with T-MENDs containing luciferase-encoding pDNA for24 h. And thereafter transgene expression (luciferase activity) was performed as described in ‘Materials and Methods’ section.

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with endosome-fusogenic SUVs2 at a ratio of 1:2 (v/v) togenerate particles with a double endosome-fusogenicenvelope, which we refer to as T-MEND. Stearylatedoctaarginine (STR-R8) solution (10% mol ofendosome-fusogenic lipid) was added to the suspensionof T-MEND to modify the outer envelope with R8.

Transfection studies

HeLa were maintained in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovineserum at 37�C under an atmosphere with 5% CO2. Cellswere seeded onto 24-well plates at a density of 4� 104 cellsper well for 48 h. A 100 ml aliquot of T-MEND containing0.4mg DNA in serum-free DMEM (1.9ml) was applied,and incubated for 3 h. Then, the culture media werereplaced with a fresh medium containing a 6% serum,and additionally incubated for 21 h. For a luciferaseassay, cells were washed with 0.5ml of phosphate-bufferedsaline (PBS; pH 7.4). Reporter lysis buffer (0.2ml) wasthen added to each well to lyse the cells. The cell suspen-sion was next frozen by placing them in an �80�C freezerfor 30min, and thawed, followed by centrifugation at 15000 rpm for 2min. The relative light units (RLU) weremeasured using a luminometer (Bio-instrument ATTOLuminescencer AB-2200, Japan), and normalized to theprotein content using the bicinchoninic acid (BCA)protein assay (Bio-Rad).

FRET optimization

pDNA was labeled with a Mirus Label IT� CX-biotinnucleic acid labeling kit (Mirus Corp., Madison, WI,USA) with minor modifications. Shortly, the pDNA waslabeled in optimized buffer supplied in the kit, but theLabel IT solution was mixed at a 1/10 concentration ofthe manufacturer’s recommended protocol of a MirusLabel IT� CX-biotin nucleic acid labeling kit, by prelim-inarily diluting the solution with distilled water. pDNAwas incubated for 60min, and then biotin labeledpDNA was purified by ethanol precipitation. The concen-tration of biotin labeled pDNA was adjusted to 0.2 mg/ml(0.05 mM final), and then a 50 ml aliquot of the solutionwas incubated with a 10 ml aliquot of the QD-545streptavidin (0.02 mM final concentration) in HEPES(pH 7.4) buffer at room temperature for at least 30min.After QD labeling, the excess QD was purified from thelabeled DNA by passing through the Sephadex G50 minicolumn, which is equilibrated with distilled water. FRETwas optimized by partially replacing a 16DMAE-ss-PRXand 46DMAE-ss-PRX with rhodamine-DMAE-ss-PRX.The synthesis of rhodamine-DMAE-ss-PRX wasdescribed in Supplementary Data. Keeping a chargeratio constant at 5, fluorescence was measured in a realtime fluorescence photometer (JASCO, FP-750 spectro-flurometer, Tokyo, Japan), where the QDs were excitedat 340 nm and an emission spectrum was collected from550 to 650 nm.

Confocal image studies

For confocal image studies 5� 104 cells/well were seededon 35-mm glass base dishes (IWAKI, Tokyo, Japan) for 2

days, and then transfected with T-MEND containing0.4 mg of QD-labeled DNA condensed with acceptor-labeled DMAE-ss-PRX at 37�C under an atmospherewith 5% CO2. At 3 h after transfection, medium waschanged with fresh culture medium, and then incubatedfor additional 3 h. The nuclei were then stained with1 mg/ml of Hoechst 33342, 15min before observation.Cells were washed with 5mM HEPES containing20U/ml of heparin sodium to remove the T-MENDbound on the cell-surface. Thereafter, medium wasreplaced with HEPES buffer (135mM NaCl, 5.4mMKCl, 1.0mM MgCl2, 1.8mM CaCl2, 5mM HEPES and10mM glucose; pH=7.3), and examined by confocallaser scanning microscopy (LSM 510 META; Carl ZeissCo. Ltd, Jena, Germany) equipped with an oil-immersionobjective lens (Plan-Apochromat 63�/NA=1.4). Nucleus(Hoechst 33342) was excited by a 2-photon Maitai laser(780 nm). The QD was then excited by light (488 nm) froman argon laser. Imaging without the use of a UV laser isadvantageous, in that it avoids cytotoxic effects of QD inresponse to the UV-radiation (19). The emitted light wasfiltered through a dichroic mirror (HFT488) and spectro-photometrically analyzed using a spectrophotometricdetector (META) over a range of 530–630 nm in 10 nmincrements. Spectral data are presented as relative fluores-cence intensity compared to maximum intensity (545 nm).

For capturing the images by spectral mode (� mode),the QD was similarly excited by light (488 nm) from anargon laser. The emitted light ranging from 537 to 569 nm,and >630 nm was simultaneously corrected by METAequipment for the QD-and rhodamine-derived signals.These data were digitally exhibited in green (G) and red(R) channels, respectively. Thereafter, the Hoechst33342signals excited by a 2-photon Maitai laser (780 nm) werecaptured, and digitally exhibited in blue (B) channel. Thethreshold for differentiating between heterochromatin andeuchromatin was determined based on the histogram ofthe fluorescence intensity in each pixel (x-axis) versus thenumber of pixel areas (y-axis). In every region of interest(r.o.i) surrounding a total nucleus, the histogram repre-sented a single-peaked spectrum at a lower level of fluor-escence intensity. The pixels with higher intensities did notexhibit any peak, since distribution of intensity in eachpixel is highly scattered. We denoted the highest tail ofthe single-peaked spectrum as a threshold fluorescent in-tensity to separate the heterochromatin and euchromatinregions.

Quantification of pDNA by means of CIDIQ method

Intra-nuclear distribution and decondensation efficiencyof plasmid DNA in intra-nuclear region was quantifiedby confocal image-assisted three-dimensionally integratedquantification (CIDIQ) method, which is originally de-veloped to quantify the distribution of exogenous DNAin endosome/lysosome, cytosol, and nucleus with sequen-tial Z-series images captured by confocal laser scanningmicroscopy (31). Z-series of images exported as 8-bitTIFF-format image data were transferred to anImage-Pro Plus software (Media Cybernetics Inc., SilverSpring, MD, USA). All of Z-series of stack files were


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opened as merged file. Then it was allowed to measurepixel area count, which satisfy the threshold pixel intensityin each RGB images or arbitrary combination of RGBchannels in marked region of interest (r.o.i.). Digitalnoise was filtered out by avoiding single-pixel signals.The counts in mock-treated cells (non-transfected cells)were negligible in the calculation of pixel areas in the Gand R channels. Detail equation is listed in SupplementaryData.

To determine the minimal number of cells required foranalysis, the statistical parameter of the coefficient of vari-ation (C.V.) was determined. When the C.V. values for thefraction of pDNA in condensation and decondensationstatus in euchromatin and heterochromatin (Fhetero

G,Fhetero

Y, FheteroR, Feu

G, FeuY and Feu

R) were plottedagainst the number of the analyzed cells, all of the C.V.values reached a plateau at 10 cells (SupplementaryFigure S1). Therefore, we concluded that 10 cells weresufficient to obtain reliable results.

RESULTS

Transgene expression transduced by T-MEND vectorcontaining pcDNA3.1-luciferase

Since decondensation kinetics in cells and nucleus arealtered depending on the pDNA condenser used (22), itsselection is one of the key determining factors for geneexpression efficiency. As the synthetic DNA condenser,we selected biocleavable polyrotaxane (DMAE-ss-PRX)in which cationic DMAE-a-CD units were linkedtogether with poly (ethylene glycol) (PEG) chains (MW;3500–4500) in monliform, and capped with disulfide-bonding (32,33) (Figure 1a). Two kinds of DMAE-ss-PRX were used in the present analysis, in which 16 or46-dimethylaminoethyl (DMAE) units were incorporatedin one molecule, in average. Here, we refer to as16DMAE-ss-PRX and 46DMAE-ss-PRX, respectively.The core formed with 16DMAE-ss-PRX represented rela-tively higher size and polydispersity index (PDI) comparedwith 46DMAE-ss-PRX and protamine (Table 1).

To deliver the pDNA/polycation core to the nucleus, weused a T-MEND, the critical structural elements of which(23) are a DNA–polycation condensed core coated withtwo nuclear membrane-fusogenic inner envelopes and twoendosome-fusogenic outer envelopes. It is designed to

overcome the endosomal membrane and nuclear mem-branes via stepwise fusion, through a ‘nuclear porecomplex (NPC)-independent pathway’ (Figure 1b). Theresulting T-MEND particles formed with 16DMAE-ss-PRX had slightly larger diameters and PDI valuescompared with those prepared using 46DMAE-ss-PRX,but the z-potential was comparable for the two samples.The size of the T-MEND prepared with protamine wassmaller, with a minimum PDI, and had a highly positive z-potential (Table 1). As shown in Figure 1c, the transfec-tion activity of the T-MEND prepared with46DMAE-ss-PRX showed a significantly higher gene ex-pression compared with those prepared with 16DMAE-ss-PRX. The mock treated cells (non-transfected cells)showed negligible luciferase activity (<103 RLU/mgprotein). Of note, the Z-potential of the T-MENDprepared with 46DMAE-ss-PRX was higher than aT-MEND prepared with protamine, a natural DNA con-denser. Thus, the stimulated transfection activity of theT-MEND prepared with 46DMAE-ss-PRX cannot beexplained by enhanced cellular uptake. As shown inFigure 1c, the transfection activity of T-MENDprepared with 46DMAE-ss-PRX represented significantlyhigher gene expression compared with those prepared with16DMAE-ss-PRX. It is unique demonstration showing animpact of modulation of condenser on transfectionactivity of T-MEND (23). In the following experiments,we compared the intra-nuclear disposition of pDNAcondensed with 16DMAE-ss-PRX and 46DMAE-ss-PRX.

Labeling of plasmid DNA and FRET optimization

For a labeling of pDNA with QD545, pDNA was firstcovalently labeled to biotin, followed by conjugationwith streptavidin-modified QD. In this labeling, weincubated the QDs with biotin-labeled pDNA at a molarratio of 0.4 (QD; 0.02 mM final and DNA; 0.05 mM final)to avoid a situation in which one pDNA possessedmultiple numbers of QDs. If the reaction proceeds stoi-chiometrically, it would be expected that the pDNA wouldcontain 0 or 1 QDs per pDNA molecule. After conjuga-tion with the QDs, QD-labeled pDNA was purified on aSephadex G50 mini column. Complete removal of freestreptavidin-labeled QD was confirmed (SupplementaryFigure S2). QD-labeling of DNA was validated by 1%agarose gel electrophoresis. As shown in Figure 2a, QDconjugated with pDNA (lane ‘Pre’) proceeded through theagarose gel more extensively compared with free QD (lane‘Q’). Free QDs are slightly charged molecules, the chargeof which is derived from surface-modified streptavidin,while pDNA is highly negatively charged due to phos-phate groups. Thus, QD, when conjugated with pDNA,developed extensively compared with free-form QDs byelectrophoresis, although the size of the QDs is smallerthan QD-conjugated pDNA. Moreover, the QD signals(Figure 2a; lane ‘Pre’) were overlapped to the pDNAsignals which are appeared by staining with ethidiumbromide (Figure 2b; lane ‘Pre’). These findings also con-firmed that QD-labeling did not occur in biotin-unlabeledpDNA (Supplementary Figure S2). Collectively, these

Table 1. Dynamic light scattering studies of the core and R8

T-MEND, using Protamine, 16DMAE-ss-PRX and 46DMAE-ss-PRX

with pcDNA 3.1-luc

Name of packaging material Size(nm)

Polydispersityindex (PDI)

Zeta-potential(mV)

Core Protamine 87–102 0.28–0.30 (+) 27–30Core 16DMAE-ss-PRX 135–150 0.24–0.37 (+) 18–23Core 46DMAE-ss-PRX 90–105 0.15–0.21 (+) 22–28T-MEND (Protamine) 150–189 0.15–0.20 (+) 42–54T-MEND (16DMAE-ss-PRX) 275–315 0.23–0.36 (+) 25–33T-MEND (46DMAE-ss-PRX) 200–221 0.15–0.19 (+) 35–43


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data indicate that QD-labeling can be achieved via specificavidin–biotin interactions. Compared with QD-unlabeledpDNA, bands corresponding to labeled pDNA (lanes‘Pre’ and ‘Pro’) were broader, probably because theband includes QD-labeled and unlabeled pDNA, asdescribed above. It is noteworthy that free form of QDwas not detected after (lane ‘Pro’) and even before (lane‘Pre’) the purification of QD-labeled pDNA. These datacollectively suggested that QDs bound to the pDNAstoichiometrically.

For FRET analysis, rhodamine-labeled PRXs werepartially replaced with non-labeled DMAE-ss-PRXkeeping a final N/P ratio at 5 (Figure 3a). To minimizea change in the condensation/decondensation profile,same length of the PEG chain (MW; 3500–4500) wasused. Each chain possessed one rhodamine unit inaverage. Acceptor-labeled polyrotaxane (Rho-DMAE-ss-PRX) was used as a portion of the condenser (5% ofits weight in 16DMAE-ss-PRX and 46DMAE-ss-PRX).This confirms that the fluorescence of rhodamine labeledon DMAE-ss-PRX is not affected when it is subjected tocondensation with QD (donor)-unlabeled pDNA, andexcited at 543 nm, the optimum wave length for its exci-tation. Thus, the effect of self-quenching is negligible inthis analysis. In both cases, partial replacement fromDMAE-ss-PRX to Rho-DMAE-ss-PRX resulted in anincrease of fluorescence intensity peaked at 590 nm byFRET, coupled with a decrease of fluorescence intensitypeaked at 530 nm. The ratio of DMAE-ss-PRX toRho-DMAE-ss-PRX was optimized where the signalsat donor and acceptor were nearly equally detected(Figure 3b and c).

Confocal imaging and CIDIQ analysis for quantificationof pDNA

The cores, in which FRET occurred between QD545-labeled pDNA and rhodamine-labeled DMAE-ss-PRXwere encapsulated to the T-MENDs, and then condensa-tion/decondensation status in nucleus was evaluated usingspectral imaging fluorescence microscopy. Typical spectraldata with a core formed with 16DMAE-ss-PRX plusRho-14DMAE-ss-PRX were represented in Figure 4

(a)

(b) (c)

Figure 3. FRET between QD-labeled pDNA and rhodamine-labeled DMAE-ss-PRX. (a) Schematic diagram illustrating the principles ofcondensation-dependent FRET occurrence between QD-labeled pDNA and rhodamine-labeled DMAE-ss-PRX. (b and c) Optimized FRETbetween QD-labeled pDNA and rhodamine-labeled DMAE-ss-PRX. FRET between QD545 (excitation at 488 nm and emission at 545 nm) andrho-DMAE-ss-PRX was monitored by spectrophotometer. For condensation, QD-labeled pDNA was condensed with 16DMAE-ss-PRX containingRho-DMAE-ss-PRX (b) and 46DMAE-ss-PRX containing Rho-DMAE-ss-PRX (c) keeping constant N/P ratio at 5.

M(a) (b)N Q Pre Pro ProM N Q Pre

EtBr(-) EtBr(+)

Free QD

QD-LabeledpDNA

1 2 3 4 5 6 7 8

19.33

7.446.224.263.472.691.881.490.93

(kbp)

Figure 2. Confirmation of QD-labeling of pDNA by agarose gel elec-trophoresis. QD and pDNA was visualized by UV-irradiation before(a) and after (b) staining with EtBr. M, size marker; lanes 1 and 5,native pDNA; lanes 2 and 6, QD only; lanes 3 and 7, QD-labeledpDNA before purification; lanes 4 and 8, QD-labeled pDNA afterpurification. QD fluorescence was detected directly under UV irradi-ation without EtBr staining (a).


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(Figure 4a for 16DMAE-ss-PRX and Figure 4c for46DMAE-ss-PRX, respectively).

Some clusters represent single-peaked spectrum at550 nm, which suggest the pDNA in completelydecondensed form. Meanwhile, some clusters representmaximum peak at 590 nm as well as 550 nm, which repre-sents pDNA in condensed form. Fluorescence rangingfrom 537 to 569 nm (donor fluorescence), and >580 nm(acceptor fluorescence) was separately collected byLSM510-META equipment, and then digitally exhibitedin green (G) and red (R) channels, respectively (Figure 4bfor 16DMAE-ss-PRX and Figure 4d for 46DMAE-ss-PRX, respectively). As described above, we conjugatedpDNA with QDs using a molar ratio of 0.4 in thisanalysis. Thus, a fraction of the pDNA contains no QDlabeling. However, this has no effect on the following dataanalysis, since pDNA, when free from donor (QD), doesnot emit fluorescence, either at 537–569 nm or at >580 nm.The nucleus was stained with Hoechst33342, and observedin a blue channel (B).

For the quantification of pDNA in condensed formand decondensed form, confocal image-assisted

three-dimensionally integrated quantification (CIDIQ)method was used which are developed to quantify theintracellular distribution of pDNA (8,31,33). Theboundaries of red and green clusters were first detectedby digitally extracting the pixels above backgroundlevels (Figure 5a). The pixels appearing as green and reddenoting pDNA in the decondensed form (Figure 5a-i),and the condensed form with (Figure 5a-ii)DMAE-ss-PRX, respectively. It is noteworthy that thepDNA detected in the cytoplasmic region (extra-nuclearregions) are mainly detected as red clusters(Supplementary Figure S3). Thus, pDNA exists in thecondensed form in the cytoplasm, presumably becauseof the protection afforded by the lipid envelope. Pixelswhich satisfy red- and green-positive clusters are definedas yellow, which represents a partially decondensedpDNA (Figure 5a-iii). Yellow pixels are barely observedin 16DMAE-ss-PRX, whereas significant amounts of pixelareas were detected in 46DMAE-ss-PRX. Similarly, theintra-nuclear region was digitally divided into heterochro-matin and euchromatin region by monitoring the fluores-cence signals of Hoechst33342 (Figure 5a-iv). The other

(a) (b)

(c) (d)

Figure 4. Optimization and intracellular visualization of FRET between QD-labeled pDNA and rhodamine-labeled DMAE-ss-PRX. (a and c)Typical spectra of clusters detected in nuclear region obtained after the transfection of T-MEND prepared with 16DMAE-ss-PRX plus rhodaminelabeled DMAE-ss-PRX (a) or 46DMAE-ss-PRX plus rhodamine labeled DMAE-ss-PRX (c) as the relative fluorescence intensity compared to themaximum intensity observed at �545 nm. (a) Blue, yellow and green arrows (left panel) and spectra lines (right panel) indicate the clusters, whereFRET was completely cancelled (right panel). That indicated as red arrow and line represent cluster where FRET was partially cancelled. (c) Greenarrow (left panel) and spectra line (right panel) indicate the clusters, where FRET was completely cancelled (right panel). Those indicated as red andblue arrows (left panel) and lines (right panel) represent cluster where FRET was partially cancelled. (b and d) Digital acquisition of QD- andrhodamine-derived signals. The emitted light derived from QD (ranging from 537 to 569 nm) and rhodamine (>580 nm) were corrected by METAequipment, and then exhibited in green (G) and red (R) channels, respectively. Hoechst33342 signals excited by a 2-photon Maitai laser (780 nm)were captured, and digitally exhibited in blue (B) channel. (b and d) represent a results of 16DMAE-ss-PRX and 46DMAE-ss-PRX, respectively.


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(a)

(b)

Figure 5. Schematic diagram illustrating the methodology to quantify the distribution and condensation/decondensation efficiency of pDNA.(a) A typical image showing the intracellular trafficking of pDNA transfected by a T-MEND prepared with 16DMAE-ss-PRX. Z-series ofconfocal images were captured by the LSM 510 META. The pixels exhibited as green, red and yellow clusters were exhibited as decondensed,condensed and partially decondensed pDNA. In addition, the heterochromatin region is defined as a region which possessed high fluorescence signalsin B channel. Typical images representing a definition of clusters which have strong signals in G and R channels were shown in a(i), a(ii), in both Gand R channels shown in a(iii) and in B channel shown in a(iv). (b) The pixel areas under the decondensed form, partially decondensed form andcondensed form in heterochromatin and euchromatin region are separately integrated through all of the Z-series of confocal images, and thendenoted as Shetero

k and Sheterok, respectively (Supplementary Equation), where k is either R or Y or G.


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intra-nuclear region was defined as the euchromatinregion. Finally, the pixel areas of red, green and yellowclusters, in euchromatin and heterochromatin regionswere independently integrated through all of the Z-seriesof confocal images, and denoted as Seu

k and Sheterok

(Figure 5b), where k denote the G or R or Y.First, pixel areas of pDNA in the whole nucleus

were compared between 16DMAE-ss-PRX and46DMAE-ss-PRX, which reflect a total nuclear deliveryof pDNA. 46DMAE-ss-PRX represents higher nucleardelivery than that of 16DMAE-ss-PRX in average,which may partially contribute to the higher gene expres-sion (Figure 6a). Subsequently, fraction of pDNA undercondensed, partially decondensed and decondensedpDNA was compared in the whole nucleus (Figure 6b).In 16DMAE-ss-PRX, nearly equal fraction of pDNAexists as decondensed and condensed form (�50% inaverage). Partially decondensed pDNA represents verylittle contribution in total nuclear pDNA. In contrast, in46DMAE-ss-PRX, decondensed pDNA mainly shearedthe pDNA in the whole nucleus (>70% in average).Of note, partially decondensed pDNA contributed asignificant part (�12% in average) of pDNA, which isone of the significant characters different form16DMAE-ss-PRX. The total pixel areas of pDNA asdecondensed form in whole nucleus were plotted inFigure 6c, which is an index for the nuclear amount ofpDNA in decondensed form. The higher value (�4-fold)

in 46DMAE-ss-PRX is in good agreement with the signifi-cantly higher gene expression in 46DMAE-ss-PRX(Figure 1c).To provide an insight into the difference in disposition

of pDNA between 16DMAE-ss-PRX and 46DMAE-ss-PRX, condensation/decondensation conditions in eu-chromatin and heterochromatin regions were independ-ently evaluated. As shown in Figure 7a, a considerableamount of pDNA was distributed to the heterochromatinand euchromatin regions in 16DMAE-ss-PRX (�60 and40%, respectively; black circle, Supplementary Table S1).However, the condensation/decondensation status ofpDNA was completely different in heterochromatin andeuchromatin. In heterochromatin, the pDNA was mainlypresent as the decondensed form, and vice versa ineuchromatin (Supplementary Table S1). As a result,decondensation efficiency in heterochromatin (Ehetero) isdrastically higher than that in euchromatin (Eeu) (�70and 25% in average, respectively; Figure 7d). On theother hand, in 46DMAE-ss-PRX, >70% of pDNA wasdistributed to euchromatin region in average (Figure 7b;black circle). Moreover, the most unique character in46DMAE-ss-PRX is that major part of pDNA (>70%)exists as a decondensed form in euchromatin region(Figure 7b and e). It should be noted here that neitherlabeling DNA with QD, nor a polycation with rhodamine,affects the size and zeta-potentials of particles(Supplementary Table S2).

(a) (b) (c)

Figure 6. Quantification of p-DNA status in whole nucleus. (a) The total pixel area of pDNA calculated as (SwholeG,+Swhole

R+SwholeY), which is an

index of the total accumulation of pDNA in the whole nucleus plotted. (b) Fraction of pDNA in condensed-, decondensed- and partiallydecondensed form in whole nucleus. Fwhole

k values were calculated as described in ‘Materials and Methods’ section, where k is R, G and Y,respectively. (c) Total pixel areas of decondensed pDNA in whole nucleus. Total pixel area of pDNA observed as decondensed form in wholenucleus was calculated as described in ‘Materials and Methods’ section, and plotted. Circles and bars represent calculated values in independent 10cells and mean values.


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DISCUSSION

In the present study, we established a method for quan-tifying the intra-nuclear localization and condensation/decondensation status of pDNA from a condenser in thenucleus. We imaged the intra-nuclear FRET, occurringfrom donor (QD)-labeled pDNA and acceptor (rhoda-mine)-labeled DMAE-ss-PRX. Thus, the definition ofFRET-cancelled signals (green) is the pDNA releasedfrom DMAE-ss-PRX, but it includes pDNA condensed

with other intra-nuclear components. Using this tech-nique, we found that modulation of the supra-molecularstructure of polyrotaxane affects intra-nuclear disposition.In our study, we evaluated intra-nuclear disposition at 3 hpost-transfection, while transient transgene expressionwas examined at 24 h post-transfection. Since the metabol-ic stability of pDNA by DNase digestion is short (half-life;<1–2 h) (34), we assume that the initial-phase of intracel-lular trafficking and intra-nuclear disposition accurately

(a)

(d) (e)

(b) (c)

Figure 7. Procedures for quantification of pDNA under condensed/decondensed form in intra-nuclear subdomains. (a and b) Fraction of pDNA incondensed-, decondensed- and partially decondensed form in nuclear subdomains. Fhetero

k and Feuk values were determined as described in ‘Materials

and Methods’ section after the transfection with 16DMAE-ss-PRX (a) and 46DMAE-ss-PRX (b), where k is R, G and Y, respectively. (c) Total pixelareas of decondensed pDNA in euchromatin region. Seu

G values were calculated as described in ‘Materials and Methods’ section, and plotted. Circlesand bars represent calculated values in independent 10 cells and mean values. After the transfection with T-MENDs prepared with 16DMAE-ss-PRX(d) and 46DMAE-ss-PRX (e), decondensation efficiencies of pDNA in whole nucleus (Ewhole), heterochromatin (Ehetero) and euchromatin (Eeu) wereplotted. Formulas for calculation of Ewhole, Ehetero and Eeu were described in Supplementary Data.


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reflect the transfection activity. The most striking differ-ence between 16DMAE-ss-PRX and 46DMAE-ss-PRX isthat pDNA cores formed with 16DMAE-ss-PRX weredominantly decondensed from polycation in heterochro-matin region (Figure 7a and d), while those prepared with46DMAE-ss-PRX were preferentially decondensed in eu-chromatin region (Figure 7b and e).

Since we previously reported that the cleavage of SSbonds in the capping structure by dithiothreitol is not suf-ficient for decondensation (35), tentative swelling andopening-up of the particle structure is not likely. We pre-viously found that the nuclear transcription efficiency oflipoplex in terms of the ratio of gene expression-positivecells to nuclear pDNA-positive cells drastically increasedin the late S and G2/M phase (36), when histone mRNAincreases. Replacement with a cationic counterpart suchas a histone might be a possible driving force fordecondensation in DMAE-ss-PRX. Taking the mechan-ism for the nuclear delivery by a T-MEND into the con-sideration, the difference in nuclear region-dependentdecondensation properties can be explained by thenuclear structure and the degree of resistance todecondensation in a cationic histone-rich environment.Histological electron microscopic observations of thenucleus clarified that the nuclear lamina meshwork playsan important role in controlling nuclear shape, and that asubset of heterochromatin is located against the innersurface of the lamina. As a result, heterochromatinforms a rim around the nucleus and is interrupted bynuclear pores (37). Therefore, it is plausible that thenascent polyplexes, introduced by nuclear membranefusion first pass through the histone-rich heterochromatinregion. Since 16DMAE-ss-PRX has less cationic DMAEunits in one chain compared to 46DMAE-ss-PRX, it mayform a looser complex. It is supported by the fact thatpDNA core formed with 16DMAE-ss-PRX are larger insize, compared to that prepared with 46DMAE-ss-PRX(Table 1 and Supplementary Table S2). Thus,decondensation of pDNA from 16DMAE-ss-PRX maybe extensively triggered in heterochromatin by replace-ment of counterpart from cationic polymer to basichistone, enriched in the heterochromatin region. Incontrast, pDNA forms more tightly condensed particlewith 46DMAE-ss-PRX. Therefore, it may be resistant tothe undesired decondensation in heterochromatin region.It remains to be clarified a driving force of the efficienteuchromatic decondensation in 46DMAE-ss-PRX.Euchromatin decondensation was subsequentlyaccelerated by the natural cellular dynamics of transcrip-tions so that the higher green pixel area was found, whichwas consistent with the respective transfection activities(Figures 1c and 7c).

Meanwhile, the issue of whether heterochromatin/eu-chromatin localization extends to the next-generationcell is an important issue to be clarified, since it isclosely linked to the sustained gene expression. However,the intra-nuclear behavior of pDNA during cell-division isnot completely understood. While some reports showedthat fluorescence-tagged pDNA is excluded fromdaughter cells (38), another report showed that pDNAstagged with protein nucleic acid (PNA) are evenly

separated between next-generation cells (39). Thequestion is whether pDNA transfected by a non-viralvector behaves as chromosomal DNA or extra-chromosomal DNA remains to be clarified in the future.The T-MEND was originally developed for use as a genecarrier for dendritic cells (23). The control of intra-nucleardisposition may offer more efficient antigen-expressionand presentation in dendritic cells and could be the basisfor practical use in DNA vaccination thereby.In conclusion, we developed a de novomethod for quan-

tifying the decondensation of pDNA in heterochromatinand euchromatin separately by means of a QD-FRETtechnique. Using this system, the findings show that thecondensation/decondensation efficacy in heterochromatinand euchromatin can be successfully controlled bymodulating the number of cationic moieties per moleculeof DMAE-ss-PRX, so that a therapeutic gene can be effi-ciently delivered to the euchromatin sub-domain. QD-FRET imaging technology is highly advantageous forclarifying the dynamics of plasmid DNA in post-nucleardelivery processes.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors thank Mr Junpei Horiuchi, Japan AdvancedInstitute of Science and Technology, for his cooperation inpreparing Rho-DMAE-ss-PRX; and Dr Milton S. Featherfor his helpful advice in writing the English article.

FUNDING

Special Coordination Funds for Promoting Science andTechnology, and in part by a Grant-in-Aid for ScientificResearch (S) and Young Scientists (A) from the Ministryof Education, Culture, Sports, Science and Technology(MEXT) of Japan. Funding for open access charge:Grant-in-Aid for Grant-in-Aid for Scientific Research(S) from the Ministry of Education, Culture, Sports,Science and Technology (MEXT) of Japan.

Conflict of interest statement. None declared.

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