Journal of Controlled Release 145 (2010) 289–296

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Journal of Controlled Release

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GENEDELIVERY

Intracellular FRET analysis of lipid/DNA complexes using flow cytometry andfluorescence imaging techniques

Sebastian Schneider a, Dominik Lenz b, Martin Holzer a, Klaus Palme c,d, Regine Süss a,⁎a Department of Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs University, D-79104 Freiburg, Germanyb Laboratório Citometria por Imagens e Biologia Molecular. Centro Universitário Vila Velha. Rua Comissário José Dantas de Melo, 21, Boa Vista, Vila Velha-ES,CEP.: 29102-770, Brazilc Institute of Biology II, Faculty of Biology, Albert-Ludwigs University, Schänzlestr. 1, D-79104 Freiburg, Germanyd Freiburg Institute of Advanced Studies (FRIAS), Centre for Biological Signalling Studies (bioss), Albert-Ludwigs University, D-79104 Freiburg, Germany

⁎ Corresponding author. Department of Pharmaceutmacy, Albert-Ludwigs University, Sonnenstr. 5, D-791047612036327; fax: +49 7612036326.

E-mail addresses: schseb47@web.de (S. Schneider),(D. Lenz), martin.holzer@pharmazie.uni-freiburg.de (Mklaus.palme@biologie.uni-freiburg.de (K. Palme),regine.suess@pharmazie.uni-freiburg.de (R. Süss).

0168-3659/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.jconrel.2010.04.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 December 2009Accepted 16 April 2010Available online 22 April 2010

Keywords:Non-viral gene therapyLipid/DNA complexesIntracellular dissociationFluorescene resonance energy transfer (FRET)Flow cytometryFluorescence microscopy

Gene therapy is a promising therapeutic concept for a large number of incurable diseases. Lipid/DNA complexes(lipoplexes) are used to deliver genes into cells. However, while large efforts have beenmade to investigate thefate of lipoplexes once inside the cell, the rate of intracellular dissociation is still largely unknown. Analysis of thedissociation rates of DNA from lipid/DNA complexes is crucial for the evaluation of a gene delivery system'sefficiency. This study introduces a new fluorescence resonance energy transfer (FRET) approach for theintracellular dissociation analysis of lipid/DNA complexes. Here, the labeling of both complex components, DNAas well as lipid, reveals whether DNA is still associated with the lipid or has dissociated. In this study the kineticproperties of complex dissociation were consistently measured with flow cytometry and fluorescencemicroscopy, and indicated that most complexes were dissociated after 24 h in A-10 cells.

ical Technology and Biophar-Freiburg, Germany. Tel.: +49

dominik.lenz@gmail.com. Holzer),

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

To date, 1537 gene therapy clinical trials have been conductedworldwide (http://www.wiley.co.uk/genetherapy/clinical). To allowthe approach of gene delivery tomake amajor contribution tomedicaltherapy, in-depth knowledge of the internalization [1] and thedissociation of gene transfer systems is required [2]. However, thereis still no reliable way to track the delivery of DNA in host cells. In2009 Ruponen et al. introduced a qRT-PCR-based quantification ofreleased DNA to analyze DNA complex dissociation [3]. Although thisis an interesting approach for the quantification of released DNA, itdoes not provide information on the intracellular localization of thetransfected DNA. Moreover, the condensation state of complexes isnot resolved, i.e. condensed, loose or unpacked complexes are notseparated.

Fluorescence resonance energy transfer (FRET) is widely acceptedas a sophisticated method for discerning spatial separation and can be

used to measure lipid/DNA condensation and dissociation process [4].In 2009 Matsumoto et al. published the first FRET-based approach forthe investigation of intracellular lipid/DNA dissociation [5]. For a FRETpair, the authors used the fluorescent dyes Cy3 and fluorescein todouble-label the DNA.

Here, we introduce a new approach for analysing the dissociationof lipid/DNA complexes by labeling both components lipid as well asDNA, and measuring FRET between them. This approach also discernsthe intracellular condensation state of the lipid/DNA complexesthemselves.

To provide an appropriate FRET pair for the intracellularinvestigation of lipid/DNA complexes, cellular autofluorescence hasto be considered. In fact, cellular autofluorescence is higher in the blueand green spectral regions than in the red region of the spectrum.Hence, by using longer wavelength dyes e.g. Cy3 and Cy5 we are ableto reduce the effects of cellular autofluorescence. Therefore for useinside the cell, this FRET pair is superior to the more popular FRETpairs described in the literature such as rhodamine and fluorescein[6].

The spectral characteristics of a Cy3/Cy5 FRET pair are compatiblefor use in a FACS-Calibur flow cytometer and also are accessible to theoptical set-up of a conventional laser scanning microscope usingexcitation wavelengths of 488 and 633 nm. This enables both theinvestigation of large cellular populations by flow cytometry, and thequantitative analysis of FRET signals, with which the intracellularcondensation state of lipid/DNA complexes can be discerned. In

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addition, the cellular localization of FRET signals can be analyzed bymeans of fluorescence microscopy.

2. Materials and methods

2.1. Cell culture

A-10 rat smooth muscle cells were obtained from the DSMZ(Braunschweig, Germany) and cultured in Dulbecco's modifiedEagle's medium supplemented with 20% (v/v) fetal calf serum in a37 °C incubator (humidified atmosphere containing 5% CO2).

2.2. Preparation of lipid/DNA complexes

DC 30 is a binary lipid mixture composed of DC cholesterol(3β-N-[(N′,N′-dimethylaminoethan)-carbamoyl]-cholesterol-HCL)and DOPE (dioleoylphosphatidyl ethanolamin) 3:7 (w/w) and waspurchased from Avanti Polar Lipids (Birmingham, USA).

Plasmid (eGFP-C1, BD Clontech, Heidelberg, Germany) wasisolated from Escherichia coli with the Mega-Prep Kit from Qiagen(Hilden, Germany) and stored at −20 °C in TE buffer (100 mM NaCl,10 mM Tris–HCl). Purity and concentration were proven by UVabsorption at 260/280 nm.

Lipoplexes were prepared in serum-free transfection medium,consisting of 25 mM sodium chloride and 250 mM saccharose.

DNA was used at a concentration of 0.01 µg/µl and DC 30 at aconcentration of 0.08 µg/µl. An equal volume of eGFP-C1 plasmidsolution was added to a dispersion of DC 30 liposomes and gentlymixed, resulting in the formation of lipoplexes with a charge ratio of1.6:1 (cationic lipid:DNA). In cellular studies 0.5 µg DNA wasincubated per well, i.e. according to this protocol 50 µl DNA solutionwas pipetted into 50 µl lipid dispersion for the complex formation of0.5 µg DNA. Lipoplexes were used 20 min after complex formation.

The size characterization of complexes was performed by dynamiclight scattering using BI-90 Plus (BIA-Brookhaven Instruments GmbH,Austria). Where indicated, lipid/DNA complexes were dissociated invitro using Zwittergent 3-14 (n-tetradecyl-N,N-dimethyl-3-ammo-nio-1-propanesulfonate) from Merck (Bad Soden, Germany). There-fore, 100 µl Zwittergent 0.1% (v/v) in sodium acetate buffer wasadded to 400 µl complex dispersion. The sample was gently mixedand analyzed after 10 min.

2.3. Fluorescence labeling

Where indicated, eGFP-C1 plasmid was labeled with Cy3 using theLabel IT Cy3 Nucleic Acid Labeling Kit at a 1:1 reagent/weight ratioaccording to the manufacturer's instructions (MIRUS, Madison, WI,USA). The labeling densities were determined by absorbancemeasurements at 1 Cy3 molecule/40 bp DNA as described by themanufacturer. Cy3 labeled DNA was diluted in transfection medium(0.01 µg/µl).

Cy5 DOPEwas synthesized as published by Schütz et al. [7]. For thesynthesis, 3 mg of DOPE and 25 µl triethylamine were dissolved in a1.2 ml mixture of chloroform/methanol 1:1 (v/v). 1 mg of Cy5 monoNHS ester (GE Healthcare, UK) in 1 ml of methanol was added. After3 h of stirring, the dried residue was dissolved in a mixture of ethanol/n-propanol 5:2 (v/v). The organic phase was diluted 1/10 (v/v) withMillipore water including 0.1% acetic acid. The solution was applied toa reverse phase column (Merck LiChroprep) and Cy5 DOPEwas elutedwith a 60% organic phase. Afterwards, the lipid was lyophilized andstored at −20 °C.

For the preparation of Cy5 labeled lipid mixtures, Cy5 DOPE andunlabeled DOPE were dissolved in ethanol/n-propanol 5:2 (v/v). Bothsolutions were mixed, so that the indicated mass ratios of Cy5 DOPEwere achieved (0.1, 1, 10% Cy5 DOPE/DOPE). DC cholesterol wasdissolved in chloroform/methanol 3:1 (v/v) and enough DC choles-

terol was added to the Cy5 DOPE/DOPE mixture as necessary toachieve a DC 30 mixture (DC cholesterol/DOPE 3:7 (m/m)).

The organic solvents were vaporized and the lipid film was thendispersed in transfection medium (0.08 µg/µl).

Finally, Cy3 DNA and Cy5 lipid complexes were prepared asdescribed for the unlabeled mixtures. For FRET experiments, singlelabeled (Cy3 DNA or Cy5 lipid) as well as double labeled (Cy3 DNA andCy5 lipid) complexes were prepared.

2.4. Flow cytometry

For the flow cytometry experiments cells were seeded in 48-wellplates at a density of 2×104 24 h prior to the experiment. After lipoplexincubation (0.5 µg DNA/well) at 37 °C for the amount of time indicated,the cells were washed 3 times with 1 ml phosphate-buffered saline(PBS) and then harvested by treatment with trypsin/EDTA (0.05%/0.02% (w/v)) solution. The cells were then pelleted, washed in 1 ml PBSand resuspended in 0.2 ml PBS. Analysis was performed within 30 minusing a 4-color FACS-Calibur (Becton Dickinson, Heidelberg, Germany).For each sample, 1×104 gated events were collected. Themeasurementof Cy3 donor, Cy5 acceptor and Cy3/Cy5 FRET fluorescence emissionwas performed as described by Sebestyen et al. [6]:

The donor was excited at 488 nm and fluorescence emission wasdetected at 585±15 nm (FL2). If fluorescence resonance energytransfer was present, acceptor emission was additionally detectedabove 670 nm (FL3). Excitation at 488 nm and the emitted fluores-cence was spatially and temporarily separated from a second laser,which excited the acceptor at 635 nm. The emitted fluorescence wasdetected at 661±8 nm (FL4). Thus, it was possible to measure thedonor signal (488 nm/FL2), the acceptor signal (635 nm/FL4) and theFRET signal (488 nm/FL3) simultaneously for every sample. Thefluorescence intensity was displayed using a logarithmic scale andanalyzed with Cell Quest Pro software (Becton Dickinson, Heidelberg,Germany).

2.5. Fluorescence microscopy

For microscopic experiments, 8 mm coverslips from Langenbrinck(Teningen, Germany) were placed into 48-well plates (BectonDickinson, Heidelberg, Germany) and coated with autoclaved gelatine(0.2% (w/v) in PBS). A-10 cells were seeded onto these coatedcoverslips in 48-well plates at a density of 2×104 24 h prior to theexperiment. Afterwards, cells were incubated with lipoplexes (0.5 µgDNA/well) for the indicated times. The cells were then fixed with 3.7%paraformaldehyde (w/w) for 30 min at room temperature andmounted onto glass slides using MobiGlow (MoBiTec, Göttingen,Germany). Images were obtained using a Zeiss LSM 510 Meta fromCarl Zeiss, Jena, Germany with a 20×/0.75 NA objective.

In colocalization studies donor (excited at 543 nm, detected at 560–615 nm) and acceptor (excited at 633 nm, detected above 650 nm)images of a sample were acquired. Image recording and image analysiswere performedwith LSM Image Browser Rel. 4.0. Each image consistedof 1024×1024 pixels measuring 0.44×0.44 µm2 and was 8 bit. Thedegree of colocalization was determined using the public domainprogram ImageJ 1.42 (National Institutes of Health, USA)with the JaCoPplugin and indicated as Pearson's coefficient [8].

In quantitative image analysis the donor (excited at 543 nm,detected at 560–615 nm), acceptor (excited at 633 nm, detectedabove 650 nm) and FRET (excited at 543 nm, detected above 650 nm)images were taken. To increase fluorescence signals the pinhole sizewas set to 2.5 airy disks. Quantitative FRET analysis was carried outusing the CellProfiler software [9]. At least 50 cells per sample weremanually segmented (i.e. the boundaries of the cells were manuallydrawn) and integrated fluorescence intensity was measured.

In photobleaching studies experiments were arranged using LSMImage Browser Rel. 4.0. According to a certain protocol: 4 fluorescent

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images of a sample were taken with the donor and acceptor settings.Then, the acceptor was bleached with a 633 nm laser at a setting ofintensity 100% for the acousto-optic tunable filter (AOTF). Thebleaching was limited to 3–4 regions of interest including lipid/DNAcomplexes. Finally, a further 4 fluorescent images were taken. Thisprotocol was repeated 20 times or stopped when the acceptorfluorescence intensity dropped to 50%.

2.6. Fluorometric studies

The fluorescence intensities of the donor (excited at 488 nm, detectedat 560 nm), the acceptor (excited at 650 nm, detected at 670 nm) and theFRET (excited at 488 nm, detected at 670 nm) were measured using aconventional spectrofluorometer (LS 50B, Perkin-Elmer, Germany).

2.7. FRET calculations

Large numbers of different FRET calculations have been publishedin the literature.

In order to compare these results to the published data, differentFRET calculations were applied.

2.8. Sensitized FRET emission

This method determines the corrected fluorescence intensity ofthe acceptor dye (Fc) after excitation at the donor wavelength. It hasbeen previously published by Youvan et al. [10]:

Fc = I DA2 −I DA

1 S1−I DA3 S2

Different optical set-ups were applied and denoted as Ix:

I1: Donor fluorescence (excited at the donor excitation wavelengthand detected at the donor emission wavelength)I2: FRET (excited at the donor excitation wavelength and detectedat the acceptor emission wavelength)I3: Acceptor fluorescence (excited at the acceptor wavelength anddetected at the acceptor emission wavelength).

Different fluorescent samples were measured and denoted as Iy:

ID : Donor-only labeled sampleIA : Acceptor-only labeled sampleIDA : Double labeled sample.

All fluorescence intensities were background corrected by mea-suring an unlabeled sample.

All samples were corrected for spectral crosstalk and backgroundintensity:

S1 =I D2I D1

; S3 =I D3I D1

for the donor labeled sample and

S2 =I A2I A3

; S4 =I A1I A3

for the acceptor labeled sample.In the case of Cy3/Cy5 FRET, S3 and S4 could be neglected [6].The samples were not normalized to the concentration of donor or

acceptor. Therefore, Fc was only calculated for experiments withoutcells.

2.9. FRET efficiency based on acceptor fluorescence

This method has been previously published by Tron et al. [11] andis generally referred to as flow cytometric energy transfer (FCET). Theapplication of FCET allows for the measurement of intracellular FRETby flow cytometry and fluorescence microscopy [12]. The calculationof the FRET efficiency Ea according to this method refers to Fc butincludes normalization to the donor concentration:

Ea =S2ðFcÞ

α I DA1 S2 + S2ðFcÞ

:

Furthermore, a new correction factor α is included:

α =I A2 × εd × LdI D1 × εa × La

:

The α factor describes the detection sensitivity of fluorescencefrom an excited acceptor molecule with respect to the sensitivity todetect an excited donor molecule. α was determined experimentallyas published by Sebestyen et al. [6] comparing the I1 signal of a donor-only labeled sample with the I2 signal of an acceptor-only labeledsample. Both were corrected by the molar absorption coefficients ofboth dyes at the donor excitation wavelength (ε) and the labelingratios (Ld and La). It has to be noted that the εa value obtained in thiswork had to be determined at very low signal-to-noise ratios.

2.10. FRET efficiency based on donor quenching

FRET efficiency (Ed) can alternatively be calculated by measuringthe amount of donor quenching, which is caused by energy transfer tothe acceptor. This calculation is the simplest realization of a FRETmeasurement and can be performed by both flow cytometry andfluorescence microscopy [12,13]:

Ed = 1− I DA1

I D1:

2.11. Statistical analysis

Statistical significance of differences was evaluated by a two-tailedpaired Student t test.

3. Results

3.1. Characterization of Cy3 DNA/Cy5 lipid complexes

3.1.1. Size characterizationFour different labeling states of lipid/DNA complexeswere prepared,

including unlabeled, single- (Cy3 DNA or Cy5 lipid) and double labeledcomplexes. For labeling of the lipid component, 1% (w/w) Cy5 DOPEwas mixed with unlabeled DOPE.

Dynamic light scattering provides direct information about thecomplex size [14]. This analysis revealed wide size distributions asindicated by a polydispersity index between 0.2 and 0.28. The NNLSalgorithm-based multimodal size distribution analysis allows for themeasurement of heterogeneous samples and resulted in meanpopulations between 540 and 610 nm. The different complexes didnot vary much in size, although the polydispersity indices must alsobe considered. It is worth mentioning that only Cy3 single labeledcomplexes included a second population at around 200 nm.

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3.1.2. Fluorometric studiesAfter size characterization, the complexes were investigated by

fluorometric analysis. Every sample was excited at 488 nm, but onlyfor double labeled complexes, where the donor and acceptor dyeswere both present at the same time, could fluorescence emission (I2)be measured at 670 nm. After correcting for spectral crosstalk andbackground subtraction sensitized FRET emission, Fc was calculatedaccording to Youvan [10] (14.4±4) (Fig. 1A). Furthermore, thenormalized FRET efficiency Ea was determined based on the acceptorfluorescence. The calculated FRET efficiency was 67% (±3). Finally,the FRET efficiency Ed was calculated based on the quenching of thedonor signal. This value (67%±2) corresponded to the acceptor basedsignal.

While the DNA labeling ratio was held constant, the Fc, Ea and Ed ofdifferent Cy5 DOPE ratios were compared (Fig. 1B,C,D).

Themost efficient fluorescence resonance energy transfer occurred ata labeling ratio of 1% Cy5 DOPE. At higher ratios (10%) the sensitized FRETemission as well as FRET efficiency Ea decreased (Fig. 1B,D), whereas thedonor quenching Ed was still present (Fig. 1C). This indicates that higherconcentrations of the acceptor dye causedonor- aswell as self-quenching.Therefore, donorquenchingmustbe combinedwithacceptor emission forreliable FRET analysis. At lower labeling ratios (0.1%) neither sufficientsensitized FRET emission (Fig. 1B) nor efficient donor quenching wasfound (Fig. 1C). Here, the FRET efficiency Ea is on the levelwith the 1% Cy5DOPE sample as the signal is normalized (Fig. 1D).

Therefore, 1% Cy5 DOPE was chosen as the labeling ratio to be usedfor later studies.

In every case the FRET signal could be removed by treatment withzwitterionic detergents (Fig. 1E).

Fig. 1. Fluorometric FRET analysis of lipid/DNA complexes. Samples were excited at488 nm and emission detected at 670 nm. (A) Comparison of different FRETcalculations. Sensitized FRET emission Fc, acceptor-based FRET efficiency Ea anddonor-based FRET efficiency Ed of lipid/DNA complexes labeled with a Cy5 DOPE ratioof 1% (m/m) are shown. (B,C,D) Fc; Ed and Ea of different Cy5 DOPE ratios (0.1, 1, 10% m/m) are compared. (E) Sensitized FRET emission Fc of lipid/DNA complexes labeled witha Cy5 DOPE ratio of 1% (m/m) after complex disruption using Zwittergent (arrowindicates start of incubation). The results are expressed as a mean (n=3)±s.e.m.

4. Cellular applicability

4.1. Flow cytometry

After size characterization and fluorometric analysis, the samedifferentially labeled complexes were administered to A-10 cells for3 h and the cellular applicability of the Cy3/Cy5 FRET system wasinvestigated by flow cytometry. The incubated lipid/DNA complexeswere taken up by 78% of the cells (histogram plot, data not shown).After excitation at the donor wavelength, the acceptor emission wasdetected at 661 nm. As the intensity of this signal depends on theconcentration of the dyes, and therefore on the amount of cellularinternalization normalization to the concentration is required. This isonly included in the calculation of FRET efficiencies. Because of this,not Fc, but the normalized FRET efficiency Ea, was calculated based onthe amount of fluorescence signal from the acceptor (66%±2) (Fig. 2).

Ea was not calculated on a cell-by-cell basis, but on the averagepopulation values of at least 104 cells per group. The mean value of atleast 3 groups was calculated and showed a standard error of ±2%.This result corresponds to the Ea and Ed values obtained byfluorometric analysis prior to cellular incubation (Fig. 2).

Furthermore, FRET efficiency based on donor quenching (Ed) wascalculated, but resulted in higher values (77%±0.7) (p=0.06).

Both calculated FRET efficiencies, Ea and Ed, show that fluorescenceresonance energy transfer is present and measurable in cellularsystems, enabling the condensation state of intracellular lipid/DNAcomplexes to be determined. These data also show that at early timepoints (3 h), lipid/DNA complexes are highly condensed structures, asthe calculated FRET efficiencies do not significantly differ or evenexceed the in vitro fluorometric studies.

4.2. Microscopic studies

4.2.1. Quantitative image analysisAs a second method, quantitative image analysis using the

CellProfiler software was applied in order to calculate FRET efficiency.Unlabeled, single or double labeled lipid/DNA complexes wereincubated for 3 h, then cells were fixed and imaged. At least fourimages of every sample were taken and at least 50 cells analyzed.Spectral correction factors and unquenched donor signals werecalculated on average population values. Based on these populationvalues, FRET efficiencies Ed and Ea were then determined on a cell-by-cell basis. Again, Ed (71%±4) exceeded Ea (67%±10) (Fig. 2). Theresults did not significantly differ from the flow cytometric studies,even though the standard errors were higher (Ed 4% and Ea 10%). In

Fig. 2. Intracellular FRET analysis of lipid/DNA complexes. Cells were incubated withlipid/DNA complexes for 3 h. In flow cytometric studies (hatched bars) samples wereexcited at 488 nm and emission detected above 670 nm. FRET efficiencies Ed and Eawere calculated based on the mean fluorescence intensities of 1×104 cells (n=3)±s.e.m. In microscopical studies (open bars) samples were excited at 543 nm and emissiondetected above 650 nm. FRET efficiencies were calculated based on the fluorescenceintensities of N50 single cells±s.e.m. using CellProfiler software. These results arecompared to the fluorometric results (solid bars) of Fig. 1A.

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principle, quantitative analysis using CellProfiler software is conve-nient for the determination of intracellular FRET efficiencies, as itcorresponds to the flow cytometric data.

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4.3. Photobleaching studies

For acceptor photobleaching studies A-10 cells were incubatedwith donor single labeled or double labeled lipid/DNA complexes for3 h. After image acquisition several regions of interest (ROI) weredefined which contained condensed lipid/DNA complexes as provenby colocalization. Within these ROI the acceptor dye was bleachedusing the 633 nm laser at 100% AOTF, while simultaneously the courseof donor fluorescence intensity was measured. In double labeledcomplexes photodestruction of the acceptor dye stopped fluorescenceresonance energy transfer and thereby resulted in an increase of thedonor signal (Fig. 3A). Here, the initial increase of acceptorfluorescence intensity indicates that Cy5 concentration dependentquenching must be considered. However, at a working concentrationof 1% Cy5 the quenching effect is comparable low as indicated inFig. 1C.

In contrast, the donor signal remained unchanged in complexeswhich were single labeled with the donor (Fig. 3B). This controlproved that the emission characteristics of the donor dye were notchanged by bleaching, i.e. the increase of donor intensity in thepresence of a bleached acceptor dye as represented by the doublelabeled complexes must be due to break down of energy transfer.

Fluorometric, flow cytometric and microscopical studies prove thepresence of FRET in Cy3 DNA/Cy5 lipid complexes. Moreover, thecondensation state of lipid/DNA complexes can be determined insidecellular systems.

Fig. 3. Intracellular acceptor photobleaching. Cells were incubated with lipid/DNAcomplexes for 3 h. Within a single ROI the acceptor dye was bleached at 633 nm.(A) Donor (Cy3, dashed line) and acceptor (Cy5, solid line) fluorescence of doublelabeled complexes in a time series. (B) Donor and acceptor fluorescence of donor-singlelabeled complexes as a negative control in a time series.

5. Kinetics of intracellular complex dissociation

The intracellular dissociation of lipid/DNA complexes was kinet-ically investigated using the Cy3/Cy5 FRET pair at 3, 5, 9 and 24 h aftercomplex incubation had started. To measure the kinetics of intracel-lular dissociation, confocal colocalization analysis was added to thepreviously approved fluorescent methods.

5.1. Colocalization studies

A simple analytical approach for the determination of theintracellular condensation state of lipid/DNA complexes is to carryout colocalization studies. Confocal microscopy allows for the preciselocalization of the Cy3 labeled DNA and the Cy5 labeled lipidcomponent. In confocal experiments, double labeled complexeswere incubated for 3, 5, 9 and 24 h. Thereafter, the cells were fixedand imaged with a confocal microscope (Fig. 4).

At early time points (3 h) both components were extensivelycolocalized. Using ImageJ the correlation of both fluorescent signalswas determined and expressed by the Pearson's coefficient (PC 0.59).After 5 h, a Cy 5 DOPE signal appeared in the cytoplasm, which was

Fig. 4. Intracellular colocalization of Cy3 DNA and Cy5 lipid. Cells were incubated for 3,5, 9, 24 h (A,B,C,D) with double labeled complexes. Colocalization of Cy3 DNA (green)and Cy5 lipid (red) in fixed cells is represented in overlay confocal images. Expressionof GFP (green) 24 h after transfection of lipid/DNA complexes is shown in (E). Scalebar: 50 µm. The image next to A is an enlargement of the square indicated in the inset.Scale bar: 10 µm.

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not colocalized with Cy 3 DNA. After 9 h the cells started to expressGFP, but the fluorescent pattern was unchanged. After 24 h, GFPexpression had increased and the cytoplasmic Cy 5 DOPE signalwidely disappeared. In contrast, the Cy3 DNA signal remainedrelatively constant over time. Calculation of Pearson's coefficientclarified the reduction of colocalization over time (PC 3 h 0.59, PC 24 h0.16), indicating that the correlation of both fluorescence signals wasmarkedly reduced (70%). In spite of the marked reduction ofcolocalization, even after 24 h a few condensed complexes were stillpresent.

5.2. Flow cytometry

To quantify distance changes between both components to a highresolution, and thus to reveal the dissociation process of lipid/DNAcomplexes, FRET efficiencies, Ed and Ea, were determined at theindicated time points. In flow cytometric experiments, unlabeled,single and double labeled complexes were incubated.

Fig. 5A presents the acceptor-based calculation of FRET efficiency.At early time points (3 h) lipid/DNA complexes were highly

condensed, as indicated by efficient fluorescent energy transfer (67%).After 5 h, FRET efficiency was still high (58%) but permanentlydecreased (9 h, 45%), and resulted in a minimum level after 24 h(20%). At this time point approximately 10% of the cells expressedGFP, as determined by flow cytometry (histogram plot). These datashow that intracellular dissociation takes place (FRET ratio 24/3 h:30%), DNA is unpacked, enabling successful expression of the gene ofinterest. These results mirror those obtained from the confocalcolocalization studies (70% reduction of colocalization).

Fig. 5. Kinetics of intracellular complex dissociation. Cells were incubated with lipid/DNA complexes for 3, 5, 9, 24 h. (A) In flow cytometric studies samples were excited at488 nm and emission detected above 670 nm. FRET efficiencies Ed (dashed line) and Ea(solid line) were calculated based on fluorescencemeans of 1×104 cells (n=3)±s.e.m.(B) In microscopic studies samples were excited at 543 nm and emission detectedabove 650 nm. FRET efficiencies Ed (dashed line) and Ea (solid line) were calculatedbased on fluorescence intensities of N50 single cells±s.e.m. using the CellProfilersoftware.

FRET efficiencies based on the donor quenching signal resulted inhigher values (Fig. 5A) at all time points. However, a decrease in FRETover time could still be observed. These results are in poor correlationwith the acceptor-based method. The slower decrease of FRET (FRETratio 24/3 h: 79%) does not fit with those of the confocal colocaliza-tion studies.

5.3. Quantitative image analysis

As described for the flow cytometric experiments, the differentlylabeled complexeswere administered to A-10 cells for 3, 5, 9 and 24 h.Afterwards cells were fixed, imaged and their FRET efficiencies (Ed andEa) were calculated.

Acceptor-based FRET analysis of the lipid/DNA dissociationprocess is presented in Fig. 5B. Compared to the flow cytometricdata, the standard errors were higher (7.4–10.7%). The poorerstatistical reliability also impaired the determination of image-basedFRET efficiency after 24 h, as the fluorescent signals extensivelydiffered between individual cells and the available number of cellswas too low for reliable analysis.

FRET efficiencies based on the donor quenching signal resulted inhigher values (Fig. 5B) at all time points. Here, image analysis allowedfor lower standard errors (3.8–5.3%), and thus FRET efficiency for latetime points (24 h) could be reliably calculated. However, this decreaseof efficiencywas not as pronounced as for the acceptor-based analysis.

Overall, image based kinetics corresponded to the flow cytometricdata. Fig. 6 shows the correlation plot of both methods.

Quantitative image analysis using CellProfiler software correlatedwell with the FRET efficiencies obtained by flow cytometric analysis.

6. Discussion

The dissociation of lipid/DNA complexes is known to be a crucialstep for efficient gene delivery [15]. Therefore, knowledge ofintracellular dissociation of the lipid/DNA complex into its singlecompounds is required to optimize gene delivery systems. Fluores-cence resonance energy transfer (FRET) is a powerful tool for theinvestigation of changes in distance, as they occur during thedissociation process. In this work the suitability of a Cy3 DNA/Cy5lipid FRET pair for dissociation analysis was evaluated.

Most of previously published fluorometric studies are restricted toin vitro studies, because cellular applications are hampered by deadcells, cellular debris and unbound dye molecules [16]. Since flowcytometric and microscopical approaches allow for outgating of thesecellular subpopulations, and the amount of unbound dye is normallynegligible, these methods are more convenient for cellular studies[11].

Fig. 6. Correlation between flow cytometry and CellProfiler. The FRET efficiencies Ed andEa as measured by flow cytometry (Fig. 5A) and fluorescence microscopy (Fig. 5B) werecorrelated.

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The use of the long wavelength FRET pair Cy3/Cy5 allows for theapplication of flow cytometry as well as fluorescence microscopy.Moreover, the measurements take place in regions of the spectrumwhere cellular autofluorescence is relatively low [6].

Using flow cytometry, FRET efficiencies after 3 h of incubationbased on donor quenching (Ed) (77%) and on acceptor fluorescence(Ea) (67%) were determined. Both analyzes are often used in theliterature [6,11–13,17]. However, for the intracellular determinationof lipid/DNA complexes they provided different FRET efficiencies inthis study. The Ed efficiencies after a 3 h administration to cells (77%)were higher than the Ed efficiencies of complexes prior to incubation(67%) as determined by fluorometric analysis. An increase of FRETefficiencies after cellular internalization has already been described inthe literature [5]. The authors suggested that this phenomenon wasdue to a change in the structure of the complex, which was sensitivelymirrored by the double labeling of DNA. However, for the lipid/DNAlabeling approach of the present study this is not very likely. Forintracellular FRET analysis, donor quenching based calculations couldbe error-prone, as two different samples (donor single and doublelabeled complexes) are compared [13]. However, the extent of cellularuptake could also differ between both samples and impact Ed, becauseof the way it is calculated. Indeed, endocytic uptake is influenced bysize differences, which cannot be ruled out for both samplesconsidering the dynamic light scattering data (donor single labeledsamples included a population of smaller particles, which was notfound in the double labeled complexes). Moreover, this method lackscomprehensive correction for spectral crosstalk [13].

Hence, the method calculating FRET efficiency based on acceptorsignal was found to be superior in this study. The calculated FRETefficiency (67%) after short cellular incubation (3 h) is consistent withthe fluorometric results prior to incubation (67%). In principle, flowcytometry allows for FRET determination on a cell-by-cell basis, thusviewing subpopulations separately [11]. However, as it is unlikely thatthe proportion of lipid to DNA is constant in different complexes, wepreferred the calculation of whole population averages [17].

Using fluorescent microscopy, spatial and quantitative analysis of theintracellular condensation states of lipid/DNA complexes were per-formed. For spatial investigation colocalization studies were carried out.Cy3 DNA and Cy5 DOPEwere extensively colocalized after 3 h incubationtime, as expected when the calculated FRET efficiencies are considered.

Quantitative image-based analysis has great potential for theinvestigation of FRET, as it enables the localization of FRET signals.Thus, the cellular compartments where lipid/DNA dissociationactually occurs can be determined (e.g. cytoplasm, nucleus) [5]. Oneapproach for quantifying FRET is based on the increase in donorfluorescence after photobleaching of the acceptor dye [17]. In thisstudy Cy3 fluorescence did indeed increase in the double labeledcomplexes after bleaching of the acceptor, while Cy3 fluorescenceremained unchanged in the single labeled complexes. However, thecalculation based on photobleaching is very sensitive to the localconcentration of fluorochromes [16]. As it is likely that the lipid/DNAratio is not constant within the different complexes, we refrainedfrom applying this method for the calculation of FRET efficiencies.

To tap the full potential of fluorescencemicroscopy for the analysisof lipid/DNA dissociation, FRET efficiencies based on donor quenching(71%) and acceptor fluorescence (67%) were calculated. FRETefficiencies calculated on fluorescent images correlated well withthose obtained using flow cytometry even though the optical settingswere inconsistent (e.g. donor excitation wavelength) even though theoptical settings were inconsistent (e.g. donor excitation wavelength).However, image based analysis is statistically less reliable because ofthe smaller number of cells investigated [16]. A platform for the highthroughput of cell image analysis would readily improve statisticalreliability. The open-source software CellProfiler which was used inthis study is one approach to measure cells in a high throughputmanner [9]. This work is a feasibility study for the application of

CellProfiler in intracellular dissociation studies. In future studies, itshould easily be possible to enlarge the number of analyzed cells.

As one example for the convenience of the Cy3 DNA/Cy5 lipid FRETsystem, lipid/DNA dissociation in A-10 cells was kinetically investi-gated. A-10 cells are efficiently transfected using DC 30 lipid/DNAcomplexes [18]. Hence, efficient intracellular dissociation can beexpected to occur in this cell line. At the early time points (3 h) lipid/DNA complexeswere still highly condensed, as indicated by high FRETefficiencies (67%) and extensive colocalization (PC 0.59). These resultsare in agreement with reports from the literature [19–22] investigat-ing the dissociation of polymer/DNA complexes. Matsumoto et al. [5]published the first report about intracellular lipid/DNA dissociation,but here the authors worked with a FRET pair where both dyes werelabelled to DNA. This approach omits FRET data at early time points, asthe sandwich structure of complexes allows for too few DNA/DNAcontacts at this time to efficiently transfer energy [5].

At later time points (5, 9 h) the degree of colocalization decreasedin the present study in parallel with the calculated FRET efficiency. Inconfocal images Cy5 DOPE was now spread all around the cytoplasmas described for LPEI/DNA complexes [20]. Expression of thetransfected GFP gene simultaneously started. After 24 h, confocalimages and calculated FRET efficiencies revealed that most intracel-lular complexes had dissociated (20%, PC 0.16). Nevertheless, a fewhighly condensed complexes were still present. Because of the wayacceptor-based FRET is calculated, negatively sensitized emissionlevels, as occur at late time points (24 h), can express highly positive Evalues (EN1) [13]. These outliers can be excluded from the cell-by-cellbased image analysis. However, here the available number of cells istoo low for reliable analysis. This is a limitation to image-basedanalysis that can be improved by more sample data and calculationsbased on population means, as was applied for the flow cytometricanalysis.

Overall, these data suggest that lipid/DNA complexes areefficiently dissociated inside A-10 cells. In this respect, nuclear entryrather than dissociation seems to be the chief determining step forlipid/DNA transfection. This conclusion is in agreement with the datapublished by Matsumoto et al. [5]. Also, Ruponen et al. publishedsimilar results for the dissociation of DOTAP/DOPE lipoplexessuggesting that most of the DNA was released from the lipidcomponent within 24 h [3]. However, they applied qRT-PCR insteadof FRET.

Furthermore, these findings indicate that intracellular dissociationof lipid/DNA can be investigated using Cy3 DNA/Cy5 DOPE. Thecombination of both fluorescence microscopy and flow cytometry iswell-correlated and has great potential for the analysis of intracellularlipid/DNA dissociation. To date, there is a lack of knowledge about theintracellular dissociation of lipid/DNA complexes. Further work usingthe methods established here will contribute to the better under-standing of lipid/DNA dissociation, and to the improvement of lipidbased gene delivery.

Acknowledgments

The authors would like to thank M. Follo, J. Adrian and WilliamTeale for critical comments on the manuscript. This study wassupported by the Excellence Initiative of the German Federal andState Governments (EXC 294) and the BMBF.

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