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Originally published In Press as doi:10.1074/jbc.M305776200 on June 14, 2003

J. Biol. Chem., Vol. 278, Issue 35, 32578-32586, August 29, 2003
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Nanoparticles of Compacted DNA Transfect Postmitotic Cells*

Ge Liu {ddagger} §, DeShan Li ¶, Murali K. Pasumarthy ¶, Tomasz H. Kowalczyk ¶, Christopher R. Gedeon ¶, Susannah L. Hyatt ¶, Jennifer M. Payne ¶, Timothy J. Miller ¶ ||, Peter Brunovskis ¶, Tamara L. Fink ¶, Osman Muhammad ¶, Robert C. Moen ¶, Richard W. Hanson {ddagger} and Mark J. Cooper ¶ **

From the {ddagger}Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and Copernicus Therapeutics, Inc., Cleveland, Ohio 44106-3052

Received for publication, June 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900–360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a ~10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Although nonviral gene transfer methods transfect dividing cells, these technologies fail to transfect most postmitotic cells (1–10), with the principal exceptions of naked DNA gene transfer into muscle (11) and large volume hydrodynamic gene transfer into liver (12, 13). In dividing cells, nuclear membrane disintegration during mitosis allows plasmid DNA to enter the nucleus prior to membrane reformation. Otherwise, the intact nuclear membrane restricts transfer of naked DNA into the nucleus. The nuclear membrane pore (NMP)1 has an internal channel diameter of 25 nm (14, 15) and does not permit naked DNA to effectively cross into the nucleus, probably due to the extended size of hydrated DNA and its negative charge density (4, 16, 17). The NMP does permit passive transfer of gold particles less than 9–10 nm in diameter and linear DNA fragments up to ~300 bp (18–22) as well as facilitated transport of proteins and small DNA segments (up to ~1 kbp) having nuclear localization signals (7, 22–28). The relative inefficiency of naked DNA, liposome-DNA complexes, and protein- and polymer-based DNA conjugates to transfect nondividing cells productively remains a significant barrier for in vivo gene therapy.

Electrostatic interactions between polycationic polymers and DNA can result in conjugates consisting of one or more molecules of DNA and a sufficient number of polycations to produce a nearly charge-neutral complex (29–31). The ratio of positive to negative charges, buffer components, polycation counterion, DNA concentration, and pH, among other variables, influence the composition, size, and shape of these DNA conjugates (29, 32). Based on specific formulation methods, we have developed compacted DNA nanoparticles that consist of one molecule of DNA and 30-mer lysine polymers substituted with polyethylene glycol (PEG); these particles have the minimum possible size for a DNA/polycation conjugate based on the partial specific volumes of the constituent components (33). Due to their small size and neutral charge density, we speculated that DNA nanoparticles might cross the nuclear pore, thereby facilitating gene transfer in nondividing cells.

To test this hypothesis, we transferred naked or compacted DNA into cells having intact nuclear membranes. Our results demonstrate that compacted DNA nanoparticles effectively transfect nondividing human cells by traversing the nuclear membrane pore. Since an overwhelming majority of target cells in patients have intact nuclear membranes during the time course of in vivo gene transfer, compacted DNA nanoparticles provide an effective platform for optimizing nonviral gene therapy.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—High performance liquid chromatography grade water (W5SK-4) was used to prepare all solutions (Fisher). Most chemicals and reagents, including trypsin type I and TRITC-dextran (average molecular mass 155 kDa), were obtained from Sigma. Femtop tips and etched glass coverslips were obtained from Eppendorf Scientific. Polylysine (CK30) was prepared using an automated solid-phase peptide synthesizer (Polypeptide Laboratories). Trifluoroacetate was the counterion of the polycation. Methoxy-PEG-maleimide (10 kDa) was purchased from Shearwater Polymers. SigmaScan® software was purchased from SPSS. Polystyrene nanosphere size standards were purchased from Duke Scientific Corp.

Cell Lines—SY5Y human neuroblastoma cells were obtained from the laboratory of Dr. Mark Israel, and HuH-7 human hepatoma cells were obtained from the American Type Culture Collection. SY5Y and HuH-7 cells were grown in Dulbecco's modified Eagle's medium/F-12 medium and RPMI 1640 medium (Invitrogen), respectively, supplemented with 10% heat-inactivated fetal calf serum, 90 µg/ml penicillin, 90 units/ml streptomycin, and 2 mM glutamine. Cells were cultured at 37 °C in 5% CO2 and passaged twice weekly.

Plasmid Construction—Plasmid pCESlucSV (6,452 bp) encodes the Photinus pyralis luciferase gene transcriptionally controlled by the cytomegalovirus (CMV) immediate early enhancer and elongation factor 1-{alpha} promoter and contains the elongation factor 1-{alpha} first intron, the RU5 translational enhancer from HTLV I, the SV40 late polyadenylation signal, and the ampicillin resistance gene. Plasmid pKCERegfpSV (4,981 bp) encodes the enhanced green fluorescence protein (EGFP) gene transcriptionally controlled by the CMV immediate early enhancer and elongation factor 1-{alpha} promoter and contains the CMV intron A, the RU5 translational enhancer, the SV40 late polyadenylation signal, and the kanamycin resistance gene. pZEGFP2.9 (2,884 bp) was prepared by inserting a blunt-ended 1,636-bp AseI/AflII fragment from pEGFP-N1 (Clontech) containing the CMV promoter/enhancer, EGFP, and late SV40 polyadenylation signal into the EcoRV site of pZeo (1,248 bp), which contains the zeomycin resistance gene and the Co1 E1 bacterial DNA replication origin. pZeo was prepared by digesting pEM7/Zeo (Invitrogen) with BspHI and XbaI, followed by treatment with Klenow and self-ligation. pZEGFP5.1 (5,147 bp) and pZEGFP9.9 (9,879 bp) were constructed by inserting the 2,263-bp ScaI fragment or the 6,995-bp StuI fragment of {lambda} DNA into the PmlI site of pZEGFP2.9, respectively. pACYC184EGFPMDV (28 kbp) was prepared by ligating the 1,640-bp AseI/AflII fragment (AflII site filled in with Klenow) from pEGFP-N1 (internal BamHI site previously destroyed by Klenow fill-in) to the 3,970-bp EcoRV/AseI fragment of pACYC184 (New England Biolabs), followed by ligation of a ~22-kbp BamHI fragment from Marek's disease virus (34) (GenBankTM accession number AF24348).

Preparation of Condensing Peptides—The purity and identity of CK30 peptide was evaluated by high pressure liquid chromatography (>95% pure), mass spectroscopy, and quantitative amino acid analysis. The peptide also was shown to be >90% in monomeric form based on fast protein liquid chromatography Resource S profile as well as a quantitative 4,4'-dithiodipyridine release assay (35). The molecular mass of 10-kDa methoxy-PEG-maleimide was confirmed by gel filtration analysis, which also indicated a low polydispersity (1.01). Approximately 88% of the PEG molecules contained functional maleimide groups, and impurities as assayed by 1H NMR were 0.30 weight %. CK30PEG10k was prepared by mixing equal molar ratios of CK30 (trifluoroacetate salt at 20–50 mg/ml in 15 ml of 0.1 M phosphate buffer, pH 7.2, with 5 mM EDTA) and Mr 10,000 methoxy-PEG-maleimide (based on maleimide reactivity) (in 15 ml of dimethyl sulfoxide) at room temperature overnight. The methoxy-PEG-maleimide was added dropwise over ~5 min to the CK30 solution mixing on a Vibrax shaker. At pH 7, the reaction of maleimide with sulfhydryls proceeds at a rate 1000 times greater than its reaction with amines (36). Performance of 4,4'-dithiodipyridine release assays before and after conjugation indicated that essentially 100% of CK30 became PEG-substituted. The reaction mixture was then fractionated on a Sephadex G15 column equilibrated with 0.1% trifluoroacetic acid, and fractions containing peptides based on absorbance at 220 nm were pooled and lyophilized.

Formulation of Compacted DNA Nanoparticles—0.9 ml of DNA at a concentration of 0.2 mg/ml in water was added in 100-µl aliquots to a vortexing solution of 0.1 ml of CK30PEG10k (7.1 mg/ml) in water at room temperature over ~2 min. The DNA concentration in the final solution was 0.18 mg/ml, and the end point ratio of positive to negative charges () was 2:1. The compacted DNA sample was dialyzed in either 5% dextrose or 0.9% NaCl to remove free CK30PEG10k and unreacted PEG and stored at 4 °C. For liposome transfection experiments, DNA was compacted using the identical protocol but substituting CK30PEG10k with CK30 at a concentration of 3.0 mg/ml. Compacted DNA used in this analysis met or exceeded a series of qualification parameters, including size and shape characteristics as determined from transmission electron micrographs (EMs) and other attributes determined from light scattering, gel, serum stability, and salt stability analyses. EM and static light scattering analysis demonstrated unaggregated electron-dense, ellipsoidal DNA nanoparticles. Gel analysis indicated no detectable free or degraded DNA. Resistance of CK30PEG10k-compacted DNA to nuclease digestion was assayed by incubating complexes in 75% mouse serum (37 °C for 2 h) followed by trypsin digestion (2.5% for 40 min at room temperature) to uncomplex the DNA. Comparison of band intensities of treated and nontreated DNA on gel analysis generates a stability index ratio; >95% of compacted DNA remained intact. Whereas the initial plasmid was >90% supercoiled, DNA released from compacted nanoparticles following trypsin digestion was primarily nicked; control studies indicated that trypsin was not contaminated with residual nuclease activity. Stability of CK30PEG10k-compacted DNA in physiologic saline was assayed by the rapid addition of 5 M NaCl to achieve a final concentration of 150 mM followed by sedimentation of the DNA at 3,400 x g for 1 min at room temperature; the ratio of A260 of the supernatant divided by the A260 value of the starting material was 100 ± 10%. For CK30-compacted DNA, liposomes at the indicated concentration below provide stability in saline without precipitation.

Dynamic Light Scattering and {zeta} Potential Analysis—Compacted DNA nanoparticles at a concentration of 1.2–2.1 mg/ml were evaluated using a dynamic light scattering instrument from Particle Sizing Systems, model NICOMP 380 ZLS, using a run time of 30 min. The size was calculated assuming solid particles, and the number-weighted distribution was fit to a Gaussian curve. If the {chi}2 error determination was >3, then a NICOMP algorithm was employed. The dynamic light scattering measurements were validated using a panel of 50- and 96-nm NIST-traceable latex particles from Duke Scientific. {zeta} potential measurements were run using the same instrument at a DNA concentration of 0.2 mg/ml for 10 min. Carboxylated latex microspheres from Bangs Laboratories were used to validate the {zeta} potential measurements.

Transmission Electron Microscopy and SigmaScan® Analysis—DNA samples (10 µl) were applied for 2 min to the carbon surface of 400-mesh copper electron microscope grids covered with Formvar and carbon films (Ted Pella) and then inverted over 100-µl water droplets on parafilm for 1 min. The samples were stained with uranyl acetate (0.04% in methanol) for 2 min, and then the grids were dipped in ethanol, blotted, and air-dried. Grids were examined using a JEOL-100C transmission electron microscope, and film plates were exposed to the image at a magnification of x 20,000–40,000. The microscope was calibrated to 87.5 Å using catalase crystals. The major and minor diameters of ellipsoidal condensed DNA particles (n {cong} 100–200 particles/sample) on EM images were automatically collected using SigmaScan® software. Images were calibrated (2.36 nm/pixel) using polystyrene nanosphere size standards (41, 50, 73, and 96 nm). Data are presented as scatter plots of the log of the major versus minor diameters.

Lysine Quantitation Using a Fluorescamine Assay and DNA Nano-particle Stoichiometric Analysis—Amino groups in CK30PEG10k were assayed using a fluorometric assay (37). CK30PEG10k and DNA were mixed at molar positive to negative () charge ratios (r) of 0.2–3.0, and free amino groups were assayed in the presence of excess fluorescamine (Sigma). Binding isotherms were evaluated by plotting bound r versus input r. A biphasic process was observed corresponding to polymer association at input r < 1, whereas for r > 1, increments of CK30PEG10k resulted in no additional increase in bound r. The plateau phase of bound r in this binding isotherm was used to determine the stoichiometry of positive to negative charges in fully compacted nanoparticles.

Circular Dichroism Spectroscopy—Samples of naked and compacted EGFP plasmids (2.9, 5.1, and 28 kbp) at concentrations of 0.39–0.62 mg/ml as well as polylysine at 0.64 mg/ml were analyzed using a CD spectrophotometer, model 202, from AVIV Instruments, Inc. All measurements were performed at 4 °C in a 0.1-cm cuvette.

Growth Arrest Models—SY5Y cells at a concentration of 7 x 105 cells/well were plated in a 6-well plate and allowed to attach overnight. The next day, cells were incubated in RPMI 1640 medium containing 32 µM all-trans-retinoic acid (ICN Pharmaceuticals). Medium was changed once on day 3, at which time neurite development was evident. By days 4–5, the cells were terminally differentiated and had stopped dividing, and these cells were transfected on day 7. Control log phase cells were plated at 1.4 x 106 cells/well on the day before gene transfer. Both growth arrest and log phase cultures were 60–80% confluent on the day of gene transfer. For HuH-7, cells at a concentration of 1.5 x 106 cells/35-mm dish were plated on day 0. Cells reached 100% confluence on day 1. Culture medium was changed daily for 2 days, and cells were transfected on day 3. Control log phase cells were plated at 2 x 105 cells/well the day before gene transfer. At transfection, the cells were 20% confluent.

Transfection—Cultured cells were transfected in triplicate with Lipofectin (Invitrogen) and either naked or CK30-compacted DNA using the manufacturer's protocol. Each well of SY5Y cells was transfected with 10 µl of Lipofectin and 2 µg of DNA, whereas each well of HuH-7 cells was incubated with 8 µl of Lipofectin and 2 µg of DNA. Transfection reagents in Opti-MEM (Invitrogen) were applied to cells for 4 h (HuH-7) or 5 h (SY5Y) before changing the medium. For growth-arrested SY5Y cells, the Opti-MEM transfection mixture contained 32 µM all-trans-retinoic acid. Cells were harvested 1, 2, or 3 days after gene transfer for luciferase activity using a kit from Promega. Chemiluminescence was monitored for 10 s using a Wallac Berthold model LB 9507 luminometer. Protein content in cell lysates was measured using the Bio-Rad DC method. Data are expressed as relative light units/µg of protein.

DNA Microinjection—All DNA samples were blinded prior to microinjection, cells were scored for EGFP expression, and the data were locked before releasing the blind. One day before microinjection, 400,000 HuH-7 cells were plated on an etched glass coverslip in a 35-mm tissue culture dish. Microinjections were performed using the Eppendorf Transjector 5246 and Micromanipulator 5171 Systems mounted on a Zeiss inverted microscope. Nuclear or cytoplasmic injections were performed using a Z (depth) limit option, a 0.3-s injection time, and an injection pressure of 60–100 hectopascals. The average injection volume was estimated to be 565 ± 86 (S.E.) fl based on transfer of purified luciferase enzyme into cell lysates from HuH-7 cells. Naked or CK30PEG10k-compacted DNA complexes were diluted at a final concentration ranging from 0.2 to 100 µg/ml in an injection solution composed of 5% glucose and 0.5% TRITC-dextran (average molecular mass of 155 kDa). High Mr dextran was excluded from the nucleus following a cytoplasmic injection and was coinjected with DNA to identify those cells having an intact nuclear membrane over the time course of observation. After microinjection, cells were washed gently and placed in fresh culture medium. HuH-7 cells strongly attached to the dish, and ~60–80% of cells survived microinjection. At variable times after microinjection, transmitted light and red/green fluorescent images were collected using a cooled CCD camera controlled by IPLab software. Registration, merge, and arrangement of cell images were automatically preformed by IPLab and Adobe Photoshop programs. For cytoplasmic microinjections, transgene expression was reported as the percentage of EGFP-positive (green) cells in the population of cells that had an exclusively cytoplasmic localization of TRITC-dextran; in this fashion, cells unintentionally microinjected into the nucleus and cells that divided were excluded from analysis. For nuclear microinjections, transgene expression was reported as the percentage of green cells in the population of cells that had nuclear localization of TRITC-dextran. All DNA microinjection studies were performed three times using newly formulated batches of compacted DNA for each experiment. For cytoplasmic or nuclear microinjections, typically 150–500 or 60–200 cells, respectively, were counted for each condition evaluated in a single experiment.

Statistical Analysis—Transfection results are expressed as mean ± S.D. For microinjection studies, results are presented as mean ± S.E. An unpaired, two-tailed t test was used to evaluate differences in levels of gene expression following cellular microinjections. Prior to statistical analysis, microinjection data were transformed to a Gaussian distribution using the formula arcsin({surd}x), as is appropriate for primary data tabulated as a quotient (38). For Fig. 2, nontransformed data were fit to a modified exponential association curve, and the observed rate constants were compared using an unpaired, two-tailed t test.



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  		FIG. 2.
Time course of EGFP expression in HuH-7 cells after nuclear or cytoplasmic microinjection with 520 copies of naked or CK30PEG10k-compacted pKCERegfpSV DNA. Significant differences in EGFP expression were observed comparing compacted and naked DNA following a cytoplasmic microinjection (unpaired t test). *, p2 < 0.05. These data were fit to exponential association curves to generate the following half-times of gene expression: naked cytoplasm, 17 h; compacted cytoplasm, 11 h; naked nuclear, 6.8 h; compacted nuclear, 6.3 h. Using an unpaired t test, there were no statistically significant differences when comparing association constants for compacted DNA following a cytoplasmic or nuclear injection (p2 = 0.24), naked DNA following a cytoplasmic or nuclear injection (p2 = 0.68), or compacted and naked DNA following a nuclear injection (p2 = 0.76).

 


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Transfection of Growth-arrested Human Cells
To determine whether compacted DNA nanoparticles could transfect nondividing cells, we terminally differentiated human neuroblastoma SY5Y cells by exposing them to 32 µM all-trans-retinoic acid for 6 days. All-trans-retinoic acid induces neuronal differentiation of these cells (39), and growth arrest occurs by day 4–5; at least 90% of the cells were in G1/G0 as monitored by propidium iodide staining and fluorescence-activated cell scanning analysis (data not shown). Log phase and growth-arrested SY5Y cells were transfected with liposome mixtures of either naked or compacted DNA encoding luciferase, and transgene activity was monitored for 3 days after transfection. EM images of liposome/naked DNA mixtures yielded a typical matrix of thin fibers (Fig. 1A). Liposome/compacted DNA generated ellipsoidal electron-dense structures in a bimodal distribution (Fig. 1B); some were essentially identical in size to compacted DNA alone (Fig. 1, C and D), whereas others appeared to be small aggregates of compacted DNA. Log phase SY5Y cells were efficiently transfected by both DNA preparations, with compacted DNA generating a 6–9-fold improvement in luciferase activity on days 1–3 after gene transfer (Fig. 1E). However, liposome mixtures containing naked DNA were not able to productively transfect postmitotic SY5Y cells, with marginal activity noted on each day. In marked contrast, liposome/compacted DNA mixtures were able to robustly transfect growth-arrested SY5Y cells. The ratio of luciferase activities comparing compacted versus naked DNA preparations was 6,900-fold on day 1.



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  		FIG. 1.
Compacted DNA can robustly transfect nondividing cells. A, electron micrograph of preparation of Lipofectin and naked pCESlucSV DNA in Opti-MEM. Note the extended size and complex structure of this matrix of DNA and liposomes. Bar, 200 nm. B, electron micrograph of mixture of Lipofectin and CK30-compacted pCESlucSV DNA in Opti-MEM. Electron-dense particles are evident without the complex matrix of densities noted with naked DNA/liposome mixtures. A bimodal distribution of particle sizes is observed; unimolecularly condensed DNA nanoparticles are apparent (thin arrows), whereas larger particles may be aggregates of nanoparticles (thick arrows). Bar, 100 nm. C, electron micrograph of CK30-compacted pCESlucSV DNA in water. A homogeneous population of unimolecularly condensed ellipsoidal DNA nanoparticles is observed. Bar, 100 nm. D, electron micrograph of CK30PEG10k-compacted pKCERegfpSV DNA in 150 mM NaCl. Note the homogeneous population of unaggregated electron-dense nanoparticles comparable in volume with those shown in C, although PEG substitution of CK30 results in a higher proportion of rodlike forms. These PEG-substituted nanoparticles retain essentially identical size and shape regardless of whether they are resuspended in water, 5% glucose, saline, or Opti-MEM/Lipofectin mixtures. Bar, 100 nm. E, transfection of log phase and growth-arrested SY5Y cells. Cells were growth-arrested by cultivating them for 6 days in 32 µM all-trans-retinoic acid. Log phase and growth-arrested cells were transfected using Lipofectin mixtures consisting of 2 µg of either naked or CK30-compacted pCESlucSV DNA, and luciferase activity was monitored over the next 3 days. Data are presented as relative light units (RLU)/µg of protein and are representative of over 30 separate transfection studies performed using multiple batches of compacted DNA. F, transfection of log phase and contact-inhibited cultures of HuH-7 cells with mixtures of Lipofectin and either naked or CK30-compacted pCESlucSV DNA. Cells were harvested 1 day after gene transfer. Similar results were obtained in a second experiment.

 

To broaden the context of these results, log phase and growth-arrested human hepatoma HuH-7 cells were transfected with liposome mixtures of either naked or compacted DNA (Fig. 1F). In this contact inhibition model of growth arrest, ~94% of cells were in G0/G1 by cell cycle analysis (data not shown). Comparable with results observed in SY5Y cells, naked DNA generated low levels of transfection in growth-arrested HuH-7 cells, whereas liposome/compacted DNA mixtures generated increased levels of luciferase activity, over 360-fold higher than cells transfected with liposome/naked DNA.

These enhanced transfection results in growth-arrested cells are consistent with the hypothesis that the small size of compacted DNA nanoparticles allows for passage through the NMP. Other possibilities include (i) enhanced cytoplasmic DNA uptake when cells are transfected with liposome mixtures of compacted compared with naked DNA; (ii) decreased degradation of compacted compared with naked DNA, resulting in a sustained cytoplasmic/nuclear DNA gradient; and (iii) improved cytoplasmic transport or diffusion of compacted compared with naked DNA. To distinguish among several of these possibilities, direct cytoplasmic and nuclear microinjections of naked and compacted DNA were performed.

DNA Microinjections
Kinetics of Gene Expression—To bypass the potential influence of liposomes on cellular trafficking and to control for equal numbers of DNA molecules that enter the cell, a naked or compacted 4,981-bp plasmid encoding EGFP (pKCERegfpSV) (Fig. 1D) was directly microinjected into the cytoplasm or nucleus of HuH-7 cells. For all microinjection studies, compacted DNA was formulated with PEG-substituted CK30 to prevent aggregation of the nanoparticles in solutions having a physiologic ionic strength. To determine the optimal time for monitoring EGFP expression, a time course was performed following microinjection of 520 copies (5 µg/ml) of either naked or compacted DNA (Fig. 2). For cells receiving a cytoplasmic microinjection, nuclear membrane integrity was assured by monitoring exclusion of co-injected high Mr TRITC-dextran from the nucleus. After a lag time of ~4 h, gene expression was evident by 8 h and nearly maximal by 24 h. There was a trend for a several-hour delay in gene expression following a cytoplasmic as compared with a nuclear microinjection of compacted DNA, although this difference was not statistically significant. Second, there was no difference in the time course of gene expression between naked or compacted DNA following a direct nuclear injection, indicating that polycation uncoating from DNA in the nucleus is rapid and not rate-limiting.

DNA Concentration Dependence—To better define the efficiency of gene transfer and expression, naked and compacted pKCERegfpSV DNA at concentrations ranging from 0.2 to 100 µg/ml were microinjected into the cytoplasm and nucleus, and the relative number of green cells was determined 24 h later. Based on the observed injection volume, this concentration range results in ~21–10,000 molecules of DNA per injection. As presented in Fig. 3, there was no difference in gene expression between naked and compacted DNA following a nuclear injection at any DNA concentration, and the percentage of EGFP-positive cells reached a maximal level of 70–75%. Following a cytoplasmic injection, there was an ~10-fold improvement in the percentage of EGFP-positive cells when comparing compacted with naked DNA; the extent of gene expression per cell, however, was less than the efficiency of a direct nuclear injection. To confirm that compacted DNA was gaining access to the nucleus via the central channel of the NMP, cells were coinjected with wheat germ agglutinin (WGA), a compound that blocks {alpha}-importin-mediated transit (40, 41). Following cytoplasmic microinjections of 5 µg/ml compacted DNA and a range between 50 and 1000 µg/ml WGA, no EGFP expression was observed (Fig. 4), whereas compacted DNA alone generated 9.3% positive cells at 24 h postinjection. The inhibition in EGFP expression by WGA was reversed by 250 mM NAcGlc, a sugar known to antagonize WGA-mediated channel blockade (41).



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  		FIG. 3.
Concentration dependence of EGFP expression in HuH-7 cells after nuclear or cytoplasmic microinjection with naked or CK30PEG10k-compacted pKCERegfpSV DNA. Significant differences in EGFP expression were observed when comparing compacted and naked DNA after a cytoplasmic microinjection (unpaired t test). *, p2 < 0.05; ***, p2 < 0.0005. There were no statistically significant differences in EGFP expression between compacted or naked DNA following a nuclear microinjection.

 


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  		FIG. 4.
Compacted DNA enters the nucleus via the nuclear membrane pore internal channel. The cytoplasm of HuH-7 cells was microinjected with 5 µg/ml compacted DNA (520 copies), compacted DNA and 435 µg/ml WGA, or compacted DNA, WGA, and 250 mM NAcGlc, and cells were scored for EGFP expression 12 and 24 h post-microinjection. EGFP expression was completely blocked by WGA, and this blockade was reversed by NAcGlc.

 

Size Threshold Analysis of Compacted DNA Nanoparticles
The preceding data indicate that compacted DNA is better able to transfect cells having an intact nuclear membrane than naked DNA. To evaluate whether the mechanism for this result is related to the size of DNA nanoparticles, as would be expected if compacted DNA were transiting the nuclear pore complex, ~520 copies of naked and compacted DNAs of different sizes (2.9, 5.1, 9.9, and 28 kbp) were microinjected into the cytoplasm and nucleus of HuH-7 cells. Shown in Fig. 5A are EMs of these compacted DNA complexes. To determine whether these electron-dense particles consist of a single molecule of plasmid DNA, image analysis of these micrographs was performed using SigmaScan® software. The minor and major diameters of these ellipsoidal particles was determined, and particle volumes were calculated. Shown in Fig. 5B is a histogram of these data for the 2.9-kbp EGFP plasmid; based on the partial specific volumes of DNA and lysine as well as their associated hydration volumes (33, 42, 43), the calculated anhydrous and hydrated volumes of unimolecular and bimolecular DNA complexes are shown. More than 95% of the DNA nanoparticles contain a single molecule of DNA, and similar results were observed for the larger DNA plasmids. Peak volumes for each DNA complex versus plasmid size is plotted in Fig. 5C.



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  		FIG. 5.
DNA nanoparticles consist of a single molecule of plasmid DNA. A, electron micrographs of 2.9-, 5.1-, 9.9-, and 28-kbp CK30PEG10k-compacted EGFP expression plasmids in D5W. Bar, 100 nm. B, histogram of calculated volumes of 2.9-kbp EGFP DNA nanoparticles based on Sigma Scan® analysis of electron micrographs. Major (dl) and minor (ds) diameters of these ellipsoidal particles were used to calculate particle volumes (V) using the equation V = {pi}(ds)2(dl)/6. An and Hn refer to the calculated anhydrous and hydrated volume of DNA nanoparticles based on reference values for the partial specific volume of DNA (0.5 cm3/g) (33), lysine (0.8 cm3/g) (42), and 0.60 g of H2O bound/g of DNA (33) and 0.48 g of H2O bound/g of polylysine (43). In these calculations, particles contain a 1:1 molar charge ratio of DNA and lysine. Subscripts 1 and 2 refer to one or two molecules of DNA per particle, respectively. C, linear regression analysis of peak nanoparticle volume versus plasmid size. R2 = 0.9957. D, CD spectra of CK30PEG10k polymer alone and naked and CK30PEG10k-compacted 2.9-, 5.1-, and 28-kbp EGFP expression plasmids. Background contribution of water was subtracted from all spectra.

 

To further characterize this panel of DNA nanoparticles composed of different sizes of plasmid DNA, dynamic light scattering and {zeta}-potential analyses were performed (Table I). Each complex had a {zeta}-potential close to zero. The particle size as measured by dynamic light scattering was significantly larger than the size measured by EM (Table II); this result may be due to the contribution of the PEG halo, increased solution viscosity as fairly concentrated solutions of compacted DNA were assayed, and the elongated shape of some nanoparticles, which tends to shift this population measurement to larger sizes. In contrast to dynamic light scattering measurements, we have observed that the PEG halo is not visualized in electron micrographs (Fig. 1, C and D). Next, binding titration isotherms of varying amounts of CK30PEG10k and a fixed amount of DNA were performed to estimate nanoparticle stoichiometry (Fig. 6). Plasmid DNA was quantified based on A260 determinations, and lysine content was measured using a fluorescamine-based assay (37). As listed in Table I, each complex consists of essentially equal amounts (mol) of positive and negative charges (1.1–1.2:1). Last, to further characterize the unique secondary structure of DNA nanoparticles and to confirm that each size of DNA nanoparticle had comparable conformations, we performed CD spectral analysis on naked and compacted DNAs. As shown in Fig. 5D, naked and compacted DNAs had distinct spectra, and the CD spectra of each size of compacted DNA were essentially identical. Together, these characterization studies demonstrate that DNA nanoparticles have a 1:1 positive/negative molar charge ratio, consist of essentially one molecule of plasmid DNA per complex, and that these properties apply to each size plasmid in this panel of DNA nanoparticles.


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  		TABLE I
Dynamic light scattering, {zeta} potential, and stoichiometric analysis of compacted DNA nanoparticles

 

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  		TABLE II
Electron micrograph size analysis of compacted DNA nanoparticles

 


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  		FIG. 6.
Stoichiometric analysis of compacted DNA nanoparticles. Various amounts of CK30PEG10k were mixed with a fixed amount of plasmid DNA (input charge ratio), and free lysine moieties were measured using fluorescamine. Determination of bound lysine permitted calculation of the bound molar charge ratio.

 

Shown in Fig. 7A is a log scale scattergram of the major and minor diameters of individual nanoparticles as analyzed from EMs of these complexes. The mean particle size ± S.E. is presented in Fig. 7B, and a summary of particle dimensions is presented in Table II. Each nanoparticle preparation had essentially equivalent DNA stability in serum, implying comparable stabilities in the nuclease-rich cytoplasm. The discrete size diameters and volumes of these particles permitted a quantitative analysis of their capabilities to cross the NMP. Nuclear microinjections of naked and compacted DNA of each plasmid size generated comparable expression results (Fig. 8), indicating that transcriptional efficiency did not decrease as the plasmid size increased. After cytoplasmic microinjection of compacted DNA, gene expression significantly decreased as the particle size increased, with a sharp fall in efficiency as the minor diameter of the complex approached 25 nm. All naked DNA samples generated low percentages of gene expression. The failure of compacted DNAs above a threshold minor diameter to generate efficient gene expression after a cytoplasmic microinjection indicates that the size and shape of compacted DNA particles are key parameters to optimize when transfecting nondividing cells.



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  		FIG. 7.
Scattergram of log of major (dl) and minor (ds) diameters of ellipsoidal compacted DNA nanoparticles as visualized by electron microscopy. A, the diameters of at least 90 particles from each of 2.9-, 5.1-, 9.9-, and 28-kbp EGFP expression plasmids are plotted. Particles having equal major and minor diameters have a spherical shape, as indicated by the 45° diagonal line. Also highlighted is the 25-nm grid line on the minor diameter axis. B, mean particle major and minor diameters ± S.E.

 


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  		FIG. 8.
Size dependence of nuclear pore transit. HuH-7 cells were microinjected in the nucleus or cytoplasm with ~520 copies of either naked or CK30PEG10k-compacted 2.9-, 5.1-, 9.9-, or 28-kbp EGFP expression plasmids. Following a cytoplasmic microinjection, a decrease in EGFP expression was observed as the minor particle diameter approached 25 nm. A significant difference in EGFP expression was noted when comparing compacted and naked 2.9-kbp DNA after a cytoplasmic injection (unpaired t test). **, p2 < 0.01. Statistical analysis of the difference between compacted and naked 5.1-kbp EGFP expression plasmid following a cytoplasmic injection was not possible due to a lack of expression for naked DNA. There were no statistically significant differences in EGFP expression between compacted or naked DNA following a nuclear microinjection.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that DNA nanoparticles consisting of a single molecule of DNA generate significantly enhanced transgene expression in nondividing cells as compared with naked DNA. In liposome-based transfection studies, compacted DNA generated over 100–1,000-fold greater levels of transgene activity than naked DNA (Fig. 1, F and E). In microinjection studies (Fig. 8), compacted DNA ellipsoids having an EM-determined minor diameter less than ~24–25 nm generate efficient gene expression in cells having an intact nuclear membrane. Significantly, the threshold minor diameter for successful gene expression approximates the inner diameter of the central channel of the NMP (14, 15), suggesting that structural features of the NMP complex limit DNA nanoparticle nuclear transfer. The finding that WGA blocks compacted DNA transgene expression after a cytoplasmic microinjection (Fig. 4) supports our contention that DNA nanoparticles are crossing the NMP to access the nucleus. Other factors that need to be considered in accounting for enhanced transgene expression include the presence of cytoplasmic nucleases as well as the cytoplasmic mobility of the transferred DNA. Although compacted DNA has increased resistance to nuclease digestion compared with naked DNA (44, 45), this finding alone does not explain these robust gene transfer results, since particles having a minor diameter >25 nm failed to generate significant gene expression after cytoplasmic microinjection. The mobility of compacted DNA in the cytoplasm of cells has not been studied, although it is reasonable to speculate that diffusion may be faster than supercoiled DNA based on relative differences (>25-fold) observed in water (data not shown). Distinct mechanisms of active intracellular transport of compacted and naked DNA also may be important.

In contrast to unimolecularly compacted DNA, the failure of naked plasmid DNA to effectively transfect cells having an intact nuclear membrane can be understood based on its physical properties in solution as well as its sensitivity to nuclease digestion. Although DNA has a cross-sectional diameter of ~2 nm, it has a persistence length or average linearity of ~50 nm (46), which probably limits its ability to diffuse across the ~200-nm-long NMP channel (22, 47). Based on the 0.346-nm spacing between adjacent nucleotides (48, 49), the DNA persistence length corresponds to a ~150-bp DNA segment. These physical properties correlate with the observation that linear DNA fragments up to ~300 bp can passively diffuse through the NMP (22). Moreover, plasmid DNA assumes a complex topology in solution and has restricted mobility in the cell cytoplasm. The cytoplasmic diffusion coefficients of fluorescently labeled 500-bp DNA molecules are about one-twentieth the value of the water-based measurement, and DNA molecules of >2000 bp are essentially immobile in the cytoplasm (50). Additionally, cytoplasmic nucleases can rapidly degrade naked DNA (51, 52), thereby further limiting transgene expression.

Our results underscore the importance of formulating unaggregated condensates of polycations and DNA that contain a single molecule of nucleic acid, thereby minimizing the minor cross-sectional diameter for a given size plasmid and optimizing nuclear entry. The incorporation of PEG into the design of these DNA nanoparticles prevents aggregation in physiologic saline (Fig. 1D) or serum, even at DNA concentrations up to 12 mg/ml (data not shown). Based on image analysis of EMs of compacted DNA, most of these particles contain a single molecule of DNA and represent the minimal possible volume of a plasmid DNA condensate (Fig. 5B). Although the PEG moieties are thought to be radially dispersed and extend away from the core of compacted DNA (53), they are not stained by uranyl acetate and thus are not detected by electron microscopy. A 10-kDa PEG contains ~227 ethylene oxide groups, which should contribute about 56 nm to the diameter of the compacted nanoparticle (54), and accounts for most of the difference in particle size as determined by dynamic light scattering and EM analysis (Tables I and II). However, the PEG moieties appear to be sufficiently flexible such that the functional relationship between the EM-determined minor particle diameter corresponds closely with the previously determined nuclear membrane pore inner channel diameter. Thus, it appears that PEG plays a key role in providing particle stability in physiologic saline while not contributing significantly to the functional particle diameter.

The CD spectra of compacted DNA nanoparticles are distinct from that of naked DNA (Fig. 5D), indicating differences in secondary structure. The compacted DNA spectra also are distinct from {psi}-form DNA (31), further distinguishing the structure of unimolecular and multimolecular DNA complexes. CD spectroscopy therefore provides a useful qualitative assay for structural characterization of DNA nanoparticles.

There was no time delay in transgene expression following nuclear injection of compacted compared with naked DNA. These data indicate that PEG-substituted, compacted DNA particles rapidly dissociate in the nucleus, releasing transcriptionally active plasmid. It is conceivable that nuclear enzymes that post-translationally modify histones, such as histone acetylase, may substitute positively charged {epsilon}-amino groups in lysine with neutral acetyl groups, thereby facilitating dissociation of CK30PEG10k and plasmid DNA. Compared with nuclear injection, there is a trend for a slight delay in transgene expression following cytoplasmic injection of compacted DNA (Fig. 2), which may be accounted for by cytoplasmic diffusion delays until particles reach the nucleus, transit time across the nuclear pore, and attainment of threshold levels of nuclear plasmid sufficient to yield EGFP-positive cells. Interestingly, luciferase reporter activity in mouse lung is evident at ~4 h following an intrapulmonary dose,2 indicating rapid cellular transit, nuclear uptake, and particle uncoating in vivo.

Nuclear localization signals (NLSs) target proteins for nuclear transport that are larger than the 40–60-kDa cut-off for passive nuclear pore diffusion (18–21), and active transport of up to 25–50-MDa complexes has been reported (47). Compacted 6-kbp DNA particles have a molecular mass of ~9.4 MDa, assuming that all DNA phosphate groups are bound by {epsilon}- and {alpha}-amino groups from CK30PEG10k. Targeting and binding of compacted DNA particles to the central channel of the NMP would therefore seem required to account for the successful gene transfer of this preparation in nondividing cells. This mechanism seems plausible, since both free polylysine peptides and DNA condensates incorporating polylysine bind to {alpha}-importin with essentially equal affinity as an extended SV40 large T antigen NLS in competitive enzyme-linked immunosorbent binding assays (16, 17). Moreover, we found that incorporation of extended NLS tags to the distal end of bifunctional PEG in fully compacted DNA nanoparticles failed to improve EGFP expression following a cytoplasmic microinjection (data not shown), suggesting that the polylysine component of the CK30PEG10k condensing peptide is sufficient to enable efficient nuclear pore transit. Although incorporation of PEG into the particle probably decreases the number of sterically available free lysine moieties per complex, apparently a sufficient number of lysines are available to facilitate nuclear import of compacted DNA.

An association between DNA compaction and nuclear pore transit has been suggested by other investigators. Pollard et al. (55) microinjected naked and polyethyleneimine- and polylysine-complexed DNA into the cytoplasm and nucleus of several cell types. A 3–4-fold enhancement of gene expression was observed comparing polyethyleneimine-condensed DNA with naked DNA following a cytoplasmic injection. Although enhancement of gene expression was observed only when complexes were prepared at a positive/negative charge ratio >2, a formulation strategy that may result in compacted particles containing a single molecule of DNA, the smallest complexes reported in EMs were at least 50 nm in diameter. These complexes were also prepared in physiologic saline, which should induce aggregation of these polyethyleneimine- and polylysine-condensed particles. Moreover, gene expression following a cytoplasmic injection was relatively inefficient, since no marker expression was observed until 1,000 particles were injected per cell, whereas transgene expression was evident in our studies when ~20–100 compacted particles were introduced in the cytoplasm. In other studies, Zauner et al. (56) microinjected polylysine-DNA complexes into the cytoplasm and nucleus of primary human fibroblasts. These non-PEG-substituted particles, which aggregate in salt solutions at physiologic ionic strength, were prepared in 5% dextrose but injected in 150 mM KCl. An ~2–4-fold improvement in gene expression was observed comparing condensed to naked DNA, but the aggregation status and size of these complexes were not reported. In contrast, our PEG-substituted compacted DNA particles do not aggregate in saline, are relatively stable in serum at 37 °C, and are precisely characterized with respect to stability in saline, size, and shape, thereby permitting a quantitative assessment of the structural requirements for nuclear pore transport and gene expression. At a DNA concentration of 5 µg/ml, or 520 copies per cell, our complexes generated a 10-fold improvement in gene expression compared with naked DNA (Fig. 3). Following systemic in vivo administration, however, copy numbers of plasmid DNA per cell are probably much lower. At copy numbers less than or equal to ~100/cell, no detectable marker gene expression was observed following cytoplasmic injection of naked DNA, whereas compacted DNA generated positive signals; the -fold enhancement of compacted compared with naked DNA under conditions relevant for in vivo gene transfer therefore cannot be precisely assessed, but it may be much greater than 1 log.

The present studies were conducted using compacted DNA nanoparticles formulated with CK30 or CK30PEG10k polymers associated with a trifluoroacetate counterion at the time of DNA condensation; this formulation method generates compacted DNA particles that have an ellipsoidal shape. We have previously reported that substitution of trifluoroacetate with other counterions, including acetate, bicarbonate, or chloride, generates unimolecularly condensed DNA particles having different shapes, including rods and toroids (32). Importantly, substantial differences in transgene expression have been observed when these various compositions have been administered to animals via intramuscular or intrapulmonary routes (32, 57). For example, rodlike compacted DNA nanoparticles using the acetate form of PEG-substituted CK30 are optimal for intrapulmonary doses, and this was the formulation recently utilized in a clinical trial in subjects with cystic fibrosis. These rodlike particles have a diameter of ~12–15 nm and a length of 100–150 nm; this geometry may be optimal for larger plasmids, such as the 8.2-kbp cystic fibrosis transmembrane conductance regulator expression plasmid developed for this clinical trial, since the small diameter may facilitate nuclear pore transit. Additionally, the estimated interpore distance on the nuclear membrane of human cells is calculated to be ~290 nm based on the reported surface density of ~10 nuclear pores/µm2 of nuclear membrane (58). This interpore distance is sufficiently large that any ellipsoidal or rodlike compacted DNA nanoparticle would probably associate with only one nuclear membrane pore. This size restriction may be important, since incorporation of multiple NLS tags on a single linear DNA molecule appears to inhibit nuclear uptake compared with a single NLS substitution, possibly due to simultaneous association of one NLS-substituted naked DNA molecule with multiple nuclear pores (59).

Although significantly improved compared with naked DNA, the gene transfer efficiency of small compacted DNA nanoparticles following a cytoplasmic microinjection remains 5–7-fold lower than the efficiency observed after a nuclear injection (Fig. 3). Additionally, the intensity of EGFP expression per cell is higher in those cells receiving nuclear rather than cytoplasmic microinjections. Further optimization of the nuclear pore transit efficiency may improve our current results. For example, it is possible that more effective presentation of NLS peptides in the design of compacted DNA may increase the proficiency of our gene transfer vector.

The overwhelming majority of target cells in a human patient, including tumors, have an intact nuclear membrane over the brief time period that a gene transfer reagent is intact and available for cell transfection. In prior experiments, we have expressed transgenes in surface murine bronchial epithelial cells at efficiencies that far exceed their mitotic rate, confirming that suitably sized compacted DNA nanoparticles can transfect differentiated cells directly in animals (60). Since our particles are nontoxic and do not induce significant inflammation (61, 62), we are not boosting gene transfer efficiencies by stimulating cell division. Establishment of a nontoxic, nonviral gene transfer technology that can transfect postmitotic cells addresses a major hurdle for the successful application of in vivo gene therapy.


   FOOTNOTES
 
* This work was supported by Copernicus Therapeutics, Inc. and National Institutes of Health Grant DK-25541 (to R. W. H.). R. W. H. has a significant equity interest in Copernicus Therapeutics, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Dept. of Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Back

|| Present address: Dept. of Pharmacology, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106. Back

** To whom correspondence should be addressed: Copernicus Therapeutics, Inc., 11000 Cedar Ave., Suite 145, Cleveland, OH 44106-3052. Tel.: 216-231-0227; Fax: 216-231-9477; E-mail: mcooper{at}cgsys.com.

1 The abbreviations used are: NMP, nuclear membrane pore; PEG, polyethylene glycol; TRITC, tetramethylrhodamine isothiocyanate; CK30, N-terminal Cys-Lys30 peptide; CMV, cytomegalovirus; EGFP, enhanced green fluorescence protein; CK30PEG10k, Cys-Lys30 peptide covalently linked to a 10-kDa PEG; EM, electron micrograph; WGA, wheat germ agglutinin; NLS, nuclear localization signal. Back

2 C. R. Gedeon, S. M. Oette, A. G. Ziady, T. H. Kowalczyk, T. L. Fink, S. L. Hyatt, R. C. Moen, P. B. Davis, and M. J. Cooper, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Pamela B. Davis for review of the manuscript and Dr. Nelson F. B. Phillips and Wenhua Jia for help in performing CD spectroscopy.



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