2002 - Biophysical Characterization of PEI - DNA Complexes

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Biophysical Characterization of PEI/DNA Complexes

SIRIRAT CHOOSAKOONKRIANG,1

BRIAN A. LOBO,1

GARY S. KOE,2

JANET G. KOE,2

C. RUSSELL MIDDAUGH1

1The Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047

2Valentis, Inc., Burlingame, California 94010

Received 22 October 2002; revised 24 February 2003; accepted 14 March 2003

ABSTRACT: Themain goal of thisstudy wasto determine the effects of polyethylenimine(PEI) molecular weight and structure (750 kDa, 25 kDa, 2 kDa branched, and 25 kDa

linear PEI) and the nitrogen/phosphate (N/P) molar ratio on the physical properties and

transfection efficiencies of PEI/DNA complexes. Fourier transform infrared spectroscopy

revealed that DNA remained in the B conformation when complexed to all PEIs. Unique

alterationsin thecirculardichroism spectraof DNAwere observed in thepresence of each

PEI, whereas differential scanning calorimetry measurements showed that all PEIs

examined destabilized supercoiled DNA at N/P


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reticuloendothelial system. Upon cell entry, theseagents may also instill properties that promoteendosomal escape to avoid lysosomal degradationand enhance nuclear entry.

One class of promising nonviral vector is the

polyethylenimines (PEI). PEI is a highly water-soluble, positively charged, synthetic polymer inwhich every third atom is a nitrogen that can beprotonated as well as provide a potential branch-ing point. Approximately 20% of the nitrogens ofPEI are protonated under physiological condi-tions.3,4 As a result, the polymer can change itsionization state over a broad pH range. Thecationic amines of both branched and linear PEIalso reduce the negative charge of DNA upon

complexation. This electrostatic interaction leadsto at least a partial condensation of the normallylargehydrodynamic volume of DNA.5,6 This is seen

in 25 kDa PEI polyplexes as the formation of arelatively homogenous population of toroidal par-ticles of 4060 nm in diameter.4 Branched andlinear PEIs of various molecular weights (MW) areamong the most transfection efficient nonviralvectors in vitro7 9 and in vivo.2,1012 This hightransfection efficiency is manifested in a variety oftarget organs and delivery routes.1317 The trans-fection efficiency of PEI depends on several factorsincluding polymer MW, the conformation of PEI(e.g., linear vs. branched), and the target cell type.

This efficiency may be further enhanced by the

ability of PEI to protect DNA from enzymaticdegradation.15

Despite the potential use of PEI to deliver DNA,the relationship between the properties of PEI/DNA polyplexes and their ability to transfectcells is poorly understood. Therefore, the purposeof this study is to thoroughly characterize com-plexes formed between DNA and various formsof branched (2, 25, and 750 kDa) and linear PEI(25 kDa) by a wide variety of biophysical methods.

We then use this information in an attempt tocorrelate various physical properties with trans-

fection efficiency in cell culture. To this end, thesecondary structure of DNA upon complexationwith PEI was investigated by using Fouriertransform infrared (FTIR) and circular dichroism(CD) spectroscopy. The thermal stability of plas-mid DNA complexed to PEI was studied usingdifferential scanning calorimetry (DSC). Addition-ally, the enthalpy of binding between PEI andDNA was explored using isothermal titrationcalorimetry (ITC) and light scattering studieswere performed to assess the particle sizes andzeta potentials of the complexes.

EXPERIMENTAL SECTION

Materials

Plasmid DNA pMB 290 (4.9 kbp), pMB 237(9.1 kbp), and pMB 401 (encoding firefly lucifer-ase) [all >95% supercoiled (sc)] were provided by

Valentis, Inc. (Burlingame, CA). The DNA con-centration was determined by UV absorbanceat 260 nm using an extinction coefficient of

0.02 (mg1 cm1 mL).Branched PEIs (MW 750, 25, and 2 kDa) and

linear PEI (MW 25 kDa) were obtained fromAldrich (Milwaukee, WI) and Polysciences, Inc.,(Warrington, PA), respectively. The polymerswere used without further purification.

COS-7 and CHO-K1 cells were obtained fromAmerican Type Culture Collection (Rockville,

MD). Dulbeccos modified Eagles medium, HamsF-12 medium, and phosphate buffered saline(PBS) were acquired from BioWhittaker (Walk-

ersville, MD). Opti-MEM (reduced-serum modi-fication of MEM) and trypsin-EDTA werepurchased from Gibco (Greenland, NY). Fetal

bovine serum was obtained from Atlanta Biologi-cals (Atlanta, GA). MTT [3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] wasobtained from Sigma (St. Louis, MO).

All buffer materials (piperazine, MES, cacodylicacid, phosphoric acid, BES, EDA, EPPS, PIPES,

ACES, boric acid, ethanolamine, HEPES, TES,MOPS, TEA, and Tris) were obtained from Sigma(St. Louis, MO). Nano-purified water was used toprepare buffer solutions.

Preparation of Complexes

Stock PEI and DNA solutions were preparedbefore each experiment at various molar ratiosof PEI nitrogen (N) to DNA phosphate (P) up toN/P10. The pH of the stock PEI solution wasadjusted to the desired pH using HCl. Complexformation always utilized solutions of equal

volumes with the least concentrated componentbeing added to the more concentrated one.

Samples were continuously stirred during addi-tion and equilibrated at room temperature for20 min before measurement. Complexes werefreshly prepared before each individual measure-ment. Complexes were formed in 10 mM Trisbuffer, pH 7.4 unless otherwise noted.

FTIR Spectroscopy

FTIR spectra were obtained with a Nicolet Magna-IR 560 spectrometer equipped with a mercury

PEI/DNA COMPLEXES 1711

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 8, AUGUST 2003


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cadmium telluride detector (Madison, WI). Sam-ples were measured with attenuated total reflec-tance geometry where the sample in solution wasplaced directly in the well of a ZnSe plate (effective

pathlength 12 mm). Spectra were obtained at

4-cm1

resolution under a dry air purge byaccumulation of 256 interferograms. Subtractionof the solvent (10 mM Tris buffer) was done usingthe association peak of H2O near 2200 cm

1 as areference point.18Additional data analysis includ-ed baseline correction (1804 to 904 cm1), sevenpoint Satvisky-Golay smoothing (if needed) andOmnic Peakfind software. The final DNA concen-tration of all samples was 1 mg/mL whereasthe PEI concentration was varied for individual

complexes.

CD

CD spectra were obtained using a 0.1-cm path-length rectangular cell at 258C, a Jasco J-720spectropolarimeter (Easton, MD), and were cor-rected by subtraction of buffer spectra. Spectrawere recorded from 350 to 200 nm at a scan rate of20 nm/min and a resolution of 0.5 nm. Threespectra were accumulated and averaged for eachsample. The final DNA concentration of allsamples was 50 mg/mL (1.54104 M DNAbases). The CD signal was converted to molar

ellipticity [y], deg l mole1 cm1, smoothed with

a Jasco Fast Fourier transform algorithm, andthen baseline adjusted to zero at 345 nm to correctfor a small contribution by differential lightscattering.

DSC

DSC was performed with a model 5100 Nano-DSC[Calorimetry Sciences Corporation (CSC), Amer-ican Fork, UT]. Measurements consisted of a

single scan from 0 to 1208C a t 18C/min under3 atm of pressure. All samples were prepared in

5 mM phosphate buffer, pH 7.4 and were degassedbefore measurement. Buffer exchange of DNAwas performed by dialysis using a Slide-a-lyzer10,000 MWCO dialysis cassette (Pierce, Rockford,IL) in 2.0 L buffer at 58C overnight. Baselineswere obtained by scanning with buffer in both thesample and reference cells. Samples were ana-lyzed in duplicate or triplicate. CpCalc software(CSC) was used to subtract the baseline from thesample thermogram. The data were converted tomolar heat capacity using the MW and concentra-tion of DNA (0.5 mg/mL). All experiments used

pMB237 except for complexes of linear 25 kDaPEI which used pMB290.

ITC

Calorimetric titrations of PEI into DNA wereconducted at 258C with a titration program of25 injections of 10 mL using a CSC model 4200isothermal titration calorimeter controlled byITCRun software (CSC). Titrations consisted ofan equilibration time of 300 s to establish abaseline followed by injections at 5-min intervals.Dialysis into the required buffers and degassingwere performed on all samples before use. DNAand PEI concentrations were 0.31 and 5.45 mM,

respectively. The titration was stopped afterDNA/PEI aggregation occurred concurrent witha loss of binding heat. BindworksTM 3.0 software

was used to integrate the raw data. Blank titra-tions of PEI into buffer were performed and theheats obtained were subtracted from each bindingtitration of PEI into DNA. The observed enthalpyof binding of PEI to DNA (DHobs) was calculatedfrom the average of the first five injections of thebinding titration, divided by the molar amount ofPEI added per injection.

pH Titrations of PEI

pH measurements of HCl titrations of PEI

solutions were performed using a Mettler ToledoMP220 pH meter (0.01 pH unit sensitivity) and anaccuT pH electrode. A water bath temperaturecontroller was used to maintain a sample tem-perature of 25.080.18C. Titrations consisted ofincremental additions of 0.1 N HCl into 5 mL ofPEI solution (5.0 mg/mL) prepared in water. Eachtitration was performed without prior pH adjust-ment of the PEI solution.

Particle Size and Zeta-Potential Measurement

Samples were prepared in 10 mM Tris buffer pH7.4, which was previously filtered through 0.2-mmpolysulfone filters (Gelman Science, MI). TheDNA concentration was held constant at 100 mg/mL whereas the N/P ratios of the PEI/DNA com-plexes were varied. Mean hydrodynamic dia-meters were determined by cumulant analysisusing a dynamic light scattering (DLS) instru-ment (BT 9000AT) equipped with a 50-mW HeNelaser with a 532-nm emission wavelength (Broo-khaven Instruments Corp., Holtsville, NY). Scat-tered light was monitored at 908 to the incident

1712 CHOOSAKOONKRIANG ET AL.



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beam and the mean hydrodynamic diameter wasobtained from the diffusion coefficient using theStokes-Einstein equation. Three continuous mea-surements of 1-min duration were taken for each

sample and the results averaged.

The zeta potentials of the PEI/DNA complexeswere determined by phase analysis light scatting(PALS) at a scattering angle of 158at 258C with aZeta PALS instrument (Brookhaven InstrumentsCorp.). An electric field strength between 14 and16 V/cm was used. Data were collected with 1015cycles of the electric field for each experiment andaveraged. The zeta potential was calculated fromthe measured electrophoretic mobility using theSmoluchowski approximation. The same samples

used for DLS measurements were used for zeta-potential analysis.

Transfection Studies

Preparation of complexes was conducted asdescribed above, with the exception that a solu-tion of plasmid pMB 401, encoding firefly lucifer-ase at 50 mg/mL was used. The resultingcomplexes were diluted 10-fold into Opti-MEM

just before application to cells. All cells weremaintained in 75-cm2 flasks at 378C and 5% CO2with COS-7 cells grown in Dulbeccos modifiedEagles medium with L-glutamine and 4.5 g/L

glucose and CHO-K1 cells in Hams F-12 media

with L-glutamine, each supplemented with 10%fetal bovine serum. Cells were subcultured every4 days using standard procedures with trypsin/EDTA for cell lifting. Before seeding, the cellswere trypsinized, counted, and diluted to aconcentration of approximately 80,000 cells/mL.Then 0.1 mL of this dilution was added to eachwell of a 96-well plate and the cells wereincubated in a humid 5% CO2 incubator at 378Cfor 18 20 h. Immediately before transfection, the

cells were washed once with PBS and the complex(250 ng of DNA) was added to each well. Cells

were incubated with the complexes for 5 h. Thetransfection agent was then removed and 100 mLof culture medium was added followed by afurther incubation of 48 h. A luciferase expressionassay was performed using the Luciferase AssaySystemTM from Promega (Madison, WI) followingthe manufacturers recommended protocol. Thecells were washed with PBS and 20 mL of 1X lysisbuffer was added per well. The cells were allowedto stand at room temperature for 30 min. The platewas then placed into a FluostarTM Galaxy micro-titer plate reader (BMG, Germany) equipped with

an injector. A volume of 100mL of luciferase assayreagent was added to each well immediatelybefore measurement. A luciferase standard cali-bration curve was obtained and used to convert

light units to nanograms of luciferase. The data

are reported as the mean standard error for aminimum of three to five samples per data point.

Assessment of Cytotoxicity (MTT Assay)

Both COS-7 and CHO-K1 cells were grown asdescribed in the transfection experiments. Cellswere treated with the PEI/DNA complexes (usingthe same N/P ratio used in the transfectionstudies) or PEI alone (using the same concentra-

tion of PEI present in the complexes) for 5 h. Thecomplexes or PEI were then removed and 100 mLof culture medium was added as needed for each

cell type. The cells were then incubated for anadditional 40 h at 378C. At this point, 11 mL ofMTT was added to each well and the cells wereincubated at 378C with 5% CO2for 3 h. An aliquotof 110 mL of MTTSS (10% Triton X-1000.1 NHCl in 125 mL of isopropanol) was added to eachwell and the cells were incubated at 378C over-night. The absorbance at 570 nm was measuredusing a FluorostarTM microtiter plate reader andthe percent cell viability was calculated using thefollowing equation:

% Cell viability

Absorbance of the test sample 100

Absorbance of control cells alone

1

RESULTS

Infrared Spectral Properties of PEI/DNA Complexes

In Figure 1A, representative infrared spectra ofDNA and its complexes with 750 kDa PEI areshown stacked in order of increasing N/P ratio,

with uncomplexed DNA at the bottom andPEI alone at the top. In the absence of polymer,DNA is in the B conformation as indicated bythe presence of the guanine/thymidine (G/T)carbonyl stretching band at 1714 cm1 (represen-

tative of interstrand base-pairing), an asymmetricphosphate stretching vibration at 1224 cm1,and a sugar-phosphate coupled vibration at970 cm1.19,20 Additional bands near 1328, 1281,and 897 cm1 (the latter not shown) supportthis assignment of the B conformation.21,22 DNAvibrational bands arising from base carbonyls




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(1715 cm1), the imidazole nitrogen of guanine(1492 cm1), and the phosphate backbone(1224 cm1) have been found to be particularlysensitive to changes in cationic lipid/DNA com-plexes.19,23 Interference from PEI CH2 scissoring(1471 cm1) and NH bending (1515 cm1)

vibrations, however, make it difficult to consis-tently resolve the 1492 cm1 guanine imidazole

nitrogen mode of DNA. Therefore, only the basecarbonyl and antisymmetric phosphate stretchingvibrations were monitored in this study.

Discrete changes in these two vibrationalfrequencies upon addition of PEI to DNA areshown in Figure 1BI. The frequency of the basein-plane CO double-bond vibration is plotted as afunction of N/P ratio in panels BE. Increases inthe peak frequency of this vibration are seen incomplexes formed at increasing N/P molar ratioscontaining the 2, 25, and 750 kDa branched PEIs,with the 25 kDa branched polymer producing

the largest increase. When linear PEI is used, anabrupt increase in peak position is seen only at alow N/P ratio of 0.5. The peak position of the DNAasymmetric phosphate vibration is plotted withrespect to the N/P ratio of the complex in panelsFI of Figure 1. Decreases in the value of

this vibrational frequency are observed in com-plexes containing each MW of PEI, although the

trends differ between the individual polymers. Incontrast, no change in peak position is observed forthe symmetric phosphate vibration (1089 cm1)(data not shown), which has generally been foundto be independent of DNA geometry.19

CD of PEI/DNA Complexes

CD spectroscopy was also used to monitor thesecondary structure of DNA in PEI/DNA com-plexes. PEI has no significant CD signal withinthe UV region monitored. Therefore, the observed

Figure 1. FTIR absorbance spectra of PEI/DNA complexes in solution. The spectra

were collected and processed as described in the text. The ratio of 750 kDa branched PEI

nitrogen/DNA phosphate is indicated on the left withthe exception of DNAand PEIalone

which are also indicated on the left. The spectra are all representative of complexes at

1 mg/mL DNA concentration (A). Effect of the molar ratio of PEI/DNA complexes on DNA

vibrational modes in solution. The peak positions of the DNA base carbonyl (BE) and

DNA antisymmetric phosphate stretch (FI) are plotted against the N/P ratio of PEI/DNA. The different MWs of PEI are indicated in the individual panels.




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signals arise entirely from the DNA molecules.The spectra of PEI/DNA complexes containingeach of the four different PEI polymers are shown

in Figure 2AD. All PEI/DNA complexes pre-

pared at N/P ratios between 2 and 4 formedmicroaggregates (see below, Fig. 3A) givingartifactual spectra due to precipitation and/ordifferential light scattering.24 The CD spectrumof uncomplexed plasmid DNA is typical of theB conformation consisting of a positive peak at274 nm and a negative minimum near 246 nm.25,26

Upon PEI complexation, both regions of the DNACD spectrum are altered. Generally, the spectrashow a decrease in the value of the molar ellip-

ticity of the positive band, concomitant with ared shift as the PEI fraction of the complexes is

increased. In most cases, the negative band alsoshifts in position to higher wavelengths anddecreasing molar ellipticity values with increas-ing N/P ratio. Complexes of DNA containing thelowest MW PEI display a rapid decrease in bothbands with their values near their minimum at anN/P ratio of 1 (Fig. 2A). The decrease that is seenin the intensity of these bands with 750 kDa PEI/DNA complexes is more gradual with increasingN/P ratios and is similar to that observed withcationic lipid/DNA complexes.24,25,27,28 The linearPEI/DNA complexes show a dramatic decrease in

Figure 2. Effect of PEI MW and N/P ratio on the CD spectra of DNA (pMB 290) and

PEI/DNA complexes. (A) PEI (2 kDa)/DNA complexes; (B) PEI (750 kDa)/DNA; (C) PEI

(25kDa)/DNAcomplexes; and(D) PEI(25 kDalinear)/DNA.Legend: (&)plasmid DNA;(*)N/P 0.5; (~)N/P 1; (})N/P 2; (*)N/P 4; (~)N/P 6; and (&)N/P 10.

Figure 3. Hydrodynamic size (A) and zeta potential

(B) of PEI/DNA complexes as a function of increasing

N/P molar ratio. The data represent the mean and

standard error of at least three separate measurements.




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the intensity of the positive peak at N/P 0.5 butundergo no further spectral shifts at N/P ratios>4. However, the branched 25 kDa PEI /DNAcomplexes show a gradual decrease in intensity

of the positive peak at 276 nm with a shift to

higher wavelength (282 nm) culminating at N/Pratios >6.

DSC of PEI/DNA Complexes

The thermal stability of DNA in PEI/DNA com-plexes was investigated using DSC (Fig. 4). Thethermal disruption of the sc form of the plasmidis detected as a broad transition above 908Cwhereas the linear and nicked open circular (lin/

oc) species produce a series of small transitionswithin the 608908C range.29 The 2 and 750 kDaforms of PEI stabilized the lin/oc DNA species at

N/P ratios below 2:1 (750 kDa) and 1:1 (2 kDa).The sc DNA in these complexes, however, wasdestabilized and its transition broadened. Colloi-dal instability of 2 kDa PEI/DNA complexes (dueto the high concentration required for DSC)precluded their investigation at N/P ratios >1.The 750 kDa PEI/DNA complexes display a

shoulder near 908C at N/P ratios above 2, possiblyresulting from the stabilization of lin/oc DNA.Complexes containing 25 kDa PEI and DNA aredistinct from all other MW PEIs tested in that

both the sc and lin/oc forms of DNA are destabi-

lized at N/P 2 (Fig. 4C). Stabilization of all formsof DNA, however, was observed in the presence ofa charge excess of this polymer. Linear 25 kDaPEI in contrast, destabilized only the sc forms ofDNA at N/P ratios 2 were notcolloidally stable and manifested large exother-mic (negative) peaks upon sc DNA disruption (notshown).

ITC

To explore any possible differences in the ener-

getics of complex formation between these PEIsand DNA, calorimetric titrations of each PEI intoDNA were performed at 258C in the presence ofbuffers of varying ionization enthalpy, at lowionic strength (


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complexation process was limited by aggregationof the complexes (not shown). The observedbinding enthalpies of PEI (25 kDa) to DNA as afunction of pH and buffer ionization enthalpy are

shown in Figure 5A. The binding enthalpy of this

PEI to DNA is dependent on the pH and theionization enthalpy of the buffer, which suggeststhat protonation changes occur during complexformation. Other PEIs also displayed similarvariations in binding enthalpy under these con-ditions (not shown). Theoretically, the observedbinding enthalpy (DHobs) is linearly dependent onthe buffer ionization enthalpy (DHioniz). The y-intercept of such a relationship corresponds to thebuffer-independent binding enthalpy (DHo) and

the slope to the degree of protonation of the ligand(n), as defined by the following equation30:

DHobs DHo nDHioniz 2

Positiven values indicate that PEI is protonated

upon binding to DNA, whereas the negative y-intercepts correspond to favorable (exothermic)buffer-corrected enthalpies of binding. These

changes in protonation presumably result froman increase in pKaof the primary, secondary, andtertiary amine groups of PEI upon complexformation.

A plot of the experimentally determined valuesof n for each PEI versus pH (Fig. 5B) permits acomparison of the pH-dependent changes in pro-

tonation during complexation of each polymer toDNA. The 2 and 750 kDa PEIs clearly display twomaxima in this plot, which indicates that thesepolymers undergo two distinct shifts in pKa in this

pH range upon complexation with DNA. Becausethe branched PEIs have three different ionizablegroups (18, 28, and38 amines), the detection of onlytwo ionization events suggests that two of thethree protonatable groupson thePEI polymer maybe cooperatively shifting in pKa, producing a singleionization event. These two groups are probablythe primary and secondary amines of PEI. Ter-

tiary amines would experience a greater degreeof electrostatic suppression of protonation by theadjacent primary and secondary amines and

would therefore be the most acidic species.

Acid Titration of PEIs

Investigations to quantify the buffering capacityand the pKas of the amines of PEI in the free stateused acid titrations of each polymer in unbufferedsolution at 258C (Fig. 5C). The buffer capacity ofeach polymer was obtained from the reciprocal

Figure 5. (A) A representative plot of the variations

in the observed binding enthalpy of PEI to DNA

measured using ITC versus buffer ionization enthalpies

at 25.08C and I


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slope (d[HCl]/dpH)31 of the raw pH titration data(inset in Fig. 5C) and the pKas were thenestimated from the maxima of these buffer capa-city curves. The largest buffering capacity of all

polymers was above pH 8.0. The pKaof PEI in this

pH range decreased with increasing MW ofbranched PEI: pKa 9 for 2 kDa PEI, 8.5 forbranched and linear 25 kDa PEI, and 8.3 for750 kDa PEI. In the pH range 6.510.0, a similarbuffering capacity was observed between thelinear and branched 25 kDa PEIs. In the pHrange 4.06.0, however, a small maximum wasobserved with branched but not linear PEI. Thismaximum probably represents the average pKaof primary amines present in branched but not

linear PEI. Therefore, the large buffering capacityabove pH 7 would appear to be due to the secon-dary amines that are present in all PEIs.

DLS and Zeta-Potential Measurementof PEI/DNA Complexes

The mean hydrodynamic diameter and zeta poten-tial of PEI/DNA complexes were assessed by DLSand PALS, respectively. The average size of allPEI/DNA complexes was approximately 100 nmat the highest and lowest N/P ratios tested andindependent of PEI MW. At intermediate N/Pratios (24), an approximately two-fold increase

in size was observed with several of the polymers

(Fig. 3A). In all cases, the polydispersity of thecomplexes complex was 1mm). The zeta potentials of thesecomplexes are shown in Figure 3B. As expected,zeta potentials are negative for complexes at lowN/P ( 4).

Transfection and Cytotoxicity

The transfection efficiency and toxicity of PEI/DNA complexes were examined in COS-7 andCHO-K1 cells. With the exception of 2 kDa PEI,all PEIs showed a maximum in their dependencyof transfection efficiency on N/P molar ratio(Fig. 6). A statistical analysis of these data usinganalysis of variance found these trends in geneexpression with varying N/P ratio to be statisti-cally significant in all cases, except for the 750 kDaPEI in CHO-K1 cells and for the 2 kDa PEI in bothcell lines (Table 1). Although the 25 kDa branchedPEI produced the highest transfection efficiency

in COS-7 cells, the 25 kDa linear form showedmaximum effectiveness in CHO-K1 cells. Overall,complexes containing the 2 kDa PEI were muchless effective than any other polymer tested. Thecytotoxicity of PEI/DNA complexes and individual

PEI polymers were evaluated as a function of N/Pratio and polymer concentration by an MTT assay

(data not illustrated). Cell viability decreasedfrom 100 to 8090% with increasing N/P ratio in

both CHO-K1 and COS-7 cells. Thus, PEI/DNAcomplexes were not highly toxic under theseconditions. Complexes were approximately 10%less toxic than PEI alone.

DISCUSSION

PEI is a commonly used nonviral vector for in vitrogene delivery. Little is known, however, about

Figure 6. Transfection of COS-7 (A) and CHO-K1

cells (B) using different MWs of PEI as a function of N/P

molar ratio. Luciferase activity was measured 48 h post-

transfection. The data represent nanograms of

expressed luciferase determined as described in Experi-

mental Section. Data represent the average of 4 6

replicates and the error bars represent the standard

error of the mean.




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any possible relationship between the biophysicalproperties of PEI/DNA complexes and theirtransfection efficiencies. Therefore, the main goalof this study was to measure a wide variety ofphysical characteristics of PEI/DNA complexesand to determine if any of these propertiescorrelate within vitro transfection efficiency.

As described in previous reports, FTIR hasproven to be a practical tool for characterizing thesecondary structure of DNA in cationic lipid/DNAcomplexes.19,20,23 These reports have clearly

shown that DNA remains in the B form in cationic

lipid/DNA complexes. In the present study, weshow that DNA is also maintained in the B formwhen complexed with different amounts, MWs,and forms of PEI. We therefore conclude that theinteraction between PEI and DNA causes verylittle if any alteration to the overall helical form ofplasmid DNA. An interaction between PEI andDNA is clearly indicated, however, by the changesin position of the DNA carbonyl and asymmetricphosphate vibrations. The reduced frequency of

the asymmetric phosphate stretching vibrationcan be directly attributed to electrostatic interac-

tions between PEI and DNA. An earlier study ofcationic lipid/DNA complexes found a 3-cm1 shiftto higher frequencies. This difference is probablydue to greater dehydration at lipid/DNA ratherthan PEI/DNA interfaces. A study of PEI/DNAinteractions in multilayer films has also indicatedthat interactions occur between DNA phosphatesand the amino groups of PEI through both electro-static and hydrogen-bonding interactions.32 Theincrease in the base carbonyl vibrational frequen-cy can be interpreted as an alteration in thehydrogen bonding of this group either through

direct PEI-base interactions or through changes inthe hydration state of DNA.

The CD spectra of PEI/DNA complexes suggestsome type of change in the structure or rearrange-ment of DNA upon complexation, as shown bydiscrete changes in DNA CD peak positions andintensities. The spectra can be placed into twocategories based on spectral similarities: thosewith an N/P molar ratio 4 (an excess of PEI). It ispossible that these categories reflect an actual

rearrangement of the complex structure above and

below charge neutrality. Previous studies suggestthat these CD changes cannot be explained bydifferential scattering or absorption flatteningartifacts.24,33 A previous hypothesis that suchalterations in CD spectra arise from conversionof the DNA to C form is not supported by the FTIRresults.25,28 Collapse of the DNA into supramolec-ular chiral structures whose optical activity op-poses that of B-form DNA seems possible but hasnot been supported by detailed CD, FTIR, Raman,

and molecular dynamic studies of cationic lipid/DNA complexes24,33 which display changes in CD

spectra similar to those described here for PEI.With respect to cationic lipid/DNA complexes, itseems that these CD changes are best explained bylimited local changes in base/base interactions. Ifthese interactions between the bases decrease inthe complex, ellipticity maxima would be expectedto decrease as seen here. Previous work by Fishet al.34 and Chen et al.3537 with model systemssupports such an explanation. Thus, we believesuch local structural perturbations due perhaps toa direct interaction between PEI and the DNAbases (as reflected in the change seen in the carbo-

Table 1. Statistical Analysis of PEI Transfection by ANOVA (Analysis of Variance)

Polymer/Cell Line

Degrees of Freedom

(Effect, Error) F p Mse

COS-7

25 kDa linear (3,12) 9.843 0.0015 3.23525 kDa branched (3,16) 6.266 0.0051 28.97

750 kDa branched (3,8) 23.26 0.0003 0.4551

2 kDa branched (3,16) 1.361 0.29a 4.89 e-7

CHO-K1

25 kDa linear (3,16) 4.522 0.018 4.522

25 kDa branched (3,16) 8.362 0.0014 0.031

750 kDa branched (3,16) 2.342 0.11a 0.1358

2 kDa branched (3,16) 2.994 0.062a 3.66 e-7

aDifferences between expression levels of N/P ratios of 410 are not statistically significant(p>0.05, F1).




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nyl stretching region with FTIR) provide the mostparsimonious explanation for the observed results.

The N/P ratios at which FTIR and CD changesreach their maximal effect appear to coincide with

transfection efficiency. It seemsunlikely, however,

that the observed spectral changes in the DNAcomponent are directly related to transfection. Infact, the spectral values generally reach theirmaximal intensity or position shift at an N/P of 6or 8 and remain constant at higher ratios, whereastransfection efficiency decreases above an N/Pratio of 6. This discrepancy at high N/P ratios couldbe due to excess unbound PEI inhibiting transfec-tion by competing for binding sites on cell surfaceproteoglycans.38 Differences in transfection of

complexes containing different MW PEIs also donot correlate with the observed spectral changes.Thus, the overall spectral properties of complexes

provide an indication of the interaction betweenPEI and DNA but only indirectly relate withtransfection efficiency.

DSC studies of PEI/DNA complexes suggest aneffect of complexation on the thermal stability ofthe sc DNA component. A destabilization of scDNA was observed in complexes at low N/P ratioswith allformsof PEI. The decrease in stabilityseenat low N/P ratios probably reflects changes in DNAtertiary structure, consistent with a loss of nega-tive supercoiling of the plasmid and perhaps the

decreases in base/base interactions suggested by

theCD results. The lin/oc forms of DNA(presentasimpurities) are, in contrast, generally stabilized atlow N/P ratios. These DNA forms are not topolo-gically constrained and reveal that neutralizationof the DNA increases its thermal stability. Thecontrasting effect of PEI on the thermal stabilitiesof sc andlinear/ocDNA has also been detected withPLL but not polyarginine.29 Once again,there is noapparent direct correlation between these stabili-zation (destabilization) effects and transfection

among the various forms of PEI.The ITC titrations were able to directly demon-

stratethe protonation of PEIupon binding to DNA.The degree of protonation of PEI, however, is lowcompared with that of the helper lipid DOPE inDOTAP/DOPE liposomes.39 No apparent relation-ship exists between the different sizes of PEI andthe degree of the protonation of PEI upon com-plexation with DNA in the pH range below 7.5.

It has been proposed that the buffering of theendosome interior by PEIinduces osmotic swellingand subsequent endosomal disruption, releasingDNA for transport to the nucleus.7 For all PEIs,the region of highest buffering capacity, typically

between 8 and 9.5, lies above the physiological pHrange. Based on these physical characteristics ofPEI/DNA complexes, this hypothesis is not sup-ported. Godbey et al.40 also showed that there was

no difference in the endosomal pH in the presence

of PEI compared with the control cells. Moreover,Suh et al.3 demonstrated that unfavorable electro-static inductive effects on amine protonation occurwith increasing ionic strength under physiologicalconditions.

It is generally thought that the size and surfacecharge of gene delivery complexes are importantfactors in modulating their cellular uptake.41 Nomajor differences were observed in the size or zetapotential of complexes prepared with different

MW PEIs. Although all complexes show a similarpattern of zeta potential (inverting in sign betweenan N/P range of 2 4), maximum transfection

efficiency seems to occur near an N/P ratio of 6,arguing against any apparent correlations be-tween these parameters and activity.

We do find that the ratio of cationic polymer toDNA and the MW of PEI are factors that stronglyinfluence transfection efficiency. This is consistentwith previous results.2,11,17,4244 Two kilodaltonPEI forms complexes that do not transfect eitherCOS-7 or CHO-K1 cells. This size of PEI has shownefficient transfectionin vitro only in combinationwith replication-defective adenoviruses.45 In this

study, only positively charged complexes (>4 N/P)

successfully transfected cells. It is probable thatthese complexes require a net positive charge tofacilitate interaction with cell surface proteogly-cans, an event that seems necessary for efficienttransfection.38 Alternatively, a recent report hasfound that the majority of PEI molecules (86%)exist in the uncomplexed state in complexesprepared at N/P ratios active in transfection(presumably at N/P4).46 This finding impliesthat in vivo, if any free PEI were able to diffuse

away from the complex, the remaining particle(if initially formed at an N/P ratio of 6) would be

approximately charge neutral. This hypothesismay explain the enhanced distribution and effi-ciency of PEI complexes formed at this N/P ratioin vivo.10 Under these conditions, the toxicity of allPEIs and their DNA complexes were fairly low andtherefore do not explain the loss of transfectionefficiency at high N/P ratio.

In summary, thisstudyhas investigated severalspectral and calorimetric properties of PEI/DNAcomplexes, which do not correlate with their levelsofin vitro transfection. One limitation (althoughcurrently untested) may be the intrinsic hetero-




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geneity of these complexes in which one or moresubpopulations may be primarily responsible formost of the transfection activity. Subpopulationsof cationic lipid/DNA complexes possessing high

activity and low toxicity have been isolated using

density gradient centrifugation, suggesting thatunique compositional or structural features maybe present with these complexes.47,48 Thus, theidentification and isolation of such a populationand a biophysical comparison to the less efficientones may yield more correlative results than thosepresented here. If this effort also fails to revealcorrelations to transfection, it seems likely thatmore complex relationships between physicalproperties of complexes and transfection efficiency

may be present. Nevertheless, these biophysicalstudies much better define nonviral gene deliveryvehicles from an analytical perspective, a result of

significant pharmaceutical utility.

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2002 - Biophysical Characterization of PEI - DNA Complexes