Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4897-4902, May 1996 Genetics

Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers (//cationic lipids/cDNA) JOLANTA F. KUKOWSKA-LATALLO*, ANNA U. BIELINSKA*, JENNIFER JOHNSON*, RALPH SPINDLERt, DONALD A. TOMALIAt, AND JAMES R. BAKER, JR.*t *Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0666; and tMichigan Molecular Institute, Midland, MI 48604 Communicated by J. L. Oncley, The University of Michigan, Ann Arbor, MI, December 21, 1995 (received for review September 7, 1995)

ABSTRACT Starburst polyamidoamine dendrimers are a primary amino groups (13, 14, 15). Compared with many other new class of synthetic polymers with unique structural and types of dendritic that have recently been physical characteristics. These polymers were investigated for synthesized, PAMAM dendrimers are the only class of mac- the ability to bind DNA and enhance DNA transfer and romolecules that are unidispersed and show high charge expression in a variety of mammalian cell lines. Twenty densities restricted to the surface of the (13, 16, 17). different types of polyamidoamine dendrimers were synthe- Dendrimers have been reliably produced in large quantities sized, and the structure was confirmed using well- and can be precisely synthesized over a range of molecular defined analytical techniques. The efficiency of plasmid DNA weights similar to that of proteins. The major structural transfection using dendrimers was examined using two re- differences in PAMAM dendrimers relate to the core mole- porter gene systems: firefly luciferase and bacterial 1B-galac- cule, either ammonia (NH3) as trivalent initiator core or tosidase. The were performed using various ethylenediamine (EDA) as a tetravalent initiator core (14), dendrimers, and levels of expression of the reporter protein that starts the stepwise polymerization process and dictates were determined. Highly efficient transfection of a broad several structural characteristics of the molecule, including the range of eukaryotic cells and cell lines was achieved with overall shape, density, and surface charge. With each new layer minimal cytotoxicity using the DNA/dendrimer complexes. or generation, the molecular weight of the dendrimer more However, the ability to transfect cells was restricted to certain than doubles, and the number of surface amine groups exactly types of dendrimers and in some situations required the doubles. The defined structure and large number of surface presence of, additional compounds, such as DEAE-dextran, amino groups of PAMAM dendrimers have led to these that appeared to alter the nature of the complex. A few cell polymers being employed as a substrate for the attachment of lines demonstrated enhanced transfection with the addition of antibodies, contrast agents, and radiopharmaceuticals for ap- chloroquine, indicating endosomal localization of the com- plications in a number of different areas of biology and plexes. The capability of a dendrimer to transfect cells ap- medicine (15, 18, 19). Studies using. antibody/dendrimer con- peared to depend on the size, shape, and number of primary jugates in vitro and in vivo in experimental animals have amino groups on the surface of the polymer. However, the documented these conjugates to be nontoxic and able to target specific dendrimer most efficient in achieving transfection biologic agents to specific cells (15, 18, 19). varied between different types of cells. These studies demon- Given that PAMAM dendrimers contain defined numbers strate that Starburst dendrimers can transfect a wide variety of amino groups on the surface of the polymer that are ofcell types in vitro and offer an efficient method for producing positively charged at physiologic pH, it was hypothesized that permanently transfected cell lines. these could interact with biologically relevant poly- anions, including nucleic acids. It was further postulated that The introduction of genetic material into eukaryotic cells has DNA/dendrimer complexes might be able to transfect cells in been a major technique to investigate gene function and the a manner similar to DNA/polylysine complexes (11), but with regulation of gene expression (1, 2). In addition, recent better efficiency, given the high solubility and defined archi- advances in detecting inherited or acquired genetic disorders tecture of the dendrimer. This report details that highly have provided the possibility of transferring recombinant efficient transfection can be achieved with DNA/dendrimer genes into somatic cells to correct missing or defective gene complexes formed with certain types of dendrimers. products (3). A variety of methods have been developed to accomplish gene transfer into eukaryotic cells. These tech- niques involve the direct physical -introduction of genetic MATERIALS AND METHODS material into cells (4), the disruption of cell membranes to Dendrimer Synthesis. Ten generations of two different allow the transfer of DNA (5, 6), the use of genetically types of PAMAM dendrimers (NH3 and EDA core) were modified viruses to deliver genetic material (7, 8), and the synthesized in a stepwise process as described in detail (14). formation of DNA complexes with either inorganic salts, Each final dendrimer preparation was purified using ultrafil- polycations, or lipids to transfer the DNA across cell mem- tration and structurally characterized using a number of tech- branes (9, 10, 11). There is great utility for these techniques, niques, including electrospray ionization mass spectroscopy, but there are limitations in target cell type and in the ability to 13C and IH nuclear magnetic resonance spectroscopy, size transfer different types of genetic material (10, 11, 12). exclusion chromatography, capillary electrophoresis, HPLC, Starburst polyamidoamine (PAMAM) dendrimers are a and gel electrophoresis (14, 20, 21). Starburst PAMAM den- new class of highly branched spherical polymers that are highly drimers are identified using a standard nomenclature; for soluble in aqueous solution and have a unique surface of example, G10 (EDA) is the 10th-generation EDA core den- drimer. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in Abbreviations: PAMAM, polyamidoamine; EDA, ethylenediamine. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

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Preparation of DNA/Dendrimer Complexes. Plasmid DNA Transfections were also performed with different enhancing was amplified in bacteria and then isolated by two cycles of agents by adding the DNA/dendrimer complexes to medium cesium chloride gradient centrifugation (22). Dendrimers were that contained the additional compound(s). Compounds used diluted to an appropriate concentration in buffer containing 20 in this manner included: 0.25-1.0 ,uM DEAE-dextran chloride mM Hepes (pH 7.9), 100 mM KCl, 0.2 mM EDTA, 0.5 mM (average Mr, 500,000; Sigma), 25-100 ,uM chloroquine diphos- DTT, and 20% glycerol, with all solutions stored at 4°C. phate (Sigma), and 2-10% glycerol (Sigma). When stable DNA/dendrimer complexes were formed by incubating the clones were generated, transfections were performed with G7 two components together in a buffer containing 100 mM NaCl, (NH3) dendrimer (without dextran) and pRSV,3gal-Neo 10 mM Tris (pH 7.5), 5 mM DTT, 4% glycerol, and 1 mM and/or pRSVLuc-Neo plasmid DNA. Clones were selected by EDTA for a minimum of 5 min at room temperature. Ratios incubation in neomycin (G418, Geneticin; Life Technologies) of nucleic acid to dendrimer were based on the electrostatic at 300 ,ug/ml for 3 weeks. Control transfections were per- charge present on each component: the number of phosphate formed with three cationic lipid preparations [TFX 50 (Pro- groups in the nucleic acid compared with the number of mega), Lipofectamine, and Lipofectin (Life Technologies)] terminal NH2 groups on a dendrimer. For example, given that according to the vendor's protocols. the number of bases in 1.0 jig of DNA is 1.71 x 1015, 1.71 Analysis of the Cellular Uptake of DNA/Dendrimer Com- x 1015 negative charges are present per 1.0 jig of DNA, plexes. Linearized and dephosphorylated DNA from a 2.9-kb whereas a G7 (NH3) dendrimer has -2.65 x 1015 charges per plasmid was radiolabeled with [_y-32P]ATP using T4 polynu- ,tg. Therefore, to obtain a 1:1 charge ratio, 1.0 ,ug of DNA was cleotide kinase. After purification, 2.0 ng of radiolabeled DNA mixed with 0.65 ,tg of dendrimer. DNA/dendrimer complexes (5.2 x 107 cpm/,g) mixed with an appropriate amount of were analyzed by electrophoretic mobility-shift assays in 1% nonlabeled plasmid DNA was complexed with G7 (EDA) agarose gel in the standard Tris-borate EDTA (TBE) buffer dendrimer to achieve a 1:10 DNA-to-dendrimer charge ratio. (pH 8.3), where DNA was identified by staining with 0.1 ,ug of Rat2 cells were plated at 2.0 x 105 cells per well in six-well ethidium bromide per ml. plates and washed with serum-free medium. DNA/dendrimer Cells and Media. The cell lines used in these studies are complexes were then added and incubated with the cells for 6 listed in Table 1. All cells were maintained in standard culture h, and sampled at specific time points. The cells were washed, media (either RPMI 1640, Leibovitz L-15, or DMEM; Life processed into extracts, and separated into nuclear and mem- Technologies, Gaithersburg, MD) with 5-10% fetal calf serum brane fractions (26). Radioactivity in whole cell extract and in and 1% penicillin-streptomycin at 37°C in 5% CO2. each fraction was then quantified, and the total counts were Transfection Methods. Transfections with dendrimers were adjusted for the number of cells in the sample. performed and analyzed using expression plasmids pRSVLuc, Expression Assays. Luciferase expression was quantified at pCMVLuc, pRSVfBgal, and pCMVf3gal containing either the 24, 48, and 72 h after transfection by measuring the light firefly luciferase gene or the bacterial f3-galactosidase gene emission resulting from 1-10 ,ul Qf cell lysate incubated with under the control of either the Rous sarcoma virus or cyto- 2.35 x 10-2 ,umol of luciferin substrate (Promega; tech. bull. megalovirus long terminal repeat enhancer/promoters (23, 24, 101). Light intensity was measured in a chemiluminometer 25). One to 10 ,ug of purified plasmid DNA (per well of (LB96P; EG & G/Berthold, Gaithersburg, MD) and adjusted transfected cells) was mixed with dendrimers at a variety of for the protein concentration of the sample. The protein charge ratios, from 2:1 to 1:500. The DNA/dendrimer com- concentration in the cell lysate was measured in a standard plexes were then allowed to form for 5 min at room temper- assay (DC protein assay; Bio-Rad). Cells transfected with the ature. Six-well plates, seeded 24 h before the transfection with expression plasmid containing the j-galactosidase reporter -200,000 cells per well, were washed with serum-free medium, gene were fixed in 1.25% glutaraldehyde for 5 min at 24-72 h and 50 ,ul of the complex containing 1-10 ,ug of DNA was after transfection. Histochemical staining for B3-galactosidase added to eachvwell of cells in 1 ml of serum-free medium and expression was performed using 5-bromo-4-chloro-3-indolyl incubated for 3 h at 37°C. The medium containing the com- f3-D-galactoside (X-Gal) substrate (Life Technologies) in 5.0 plexes was then replaced with standard growth medium. The mM potassium ferrocyanide/potassium ferricyanide and 2.0 cells remained in culture for 24-72 h before being harvested mM MgCl2. The number and percentage of stained cells were for analysis of expression of the transfected plasmid DNA. determined by light microscopy. Table 1. Optimal generation of PAMAM dendrimer in transient transfection assay of different cell types and frequencies for generating stable cell lines Cell line Cell type Dendrimer Stable clone frequency Rat2 Rat embryonal fibroblast G10 1.3 x 10-3 Clone 9 Rat liver epithelium G7-G10 ND NIH/3T3 Mouse embryonal G7-G10 ND EL4 Mouse lymphoma G7 ND D5 Mouse melanoma G7 1.6 x 10-3 COS-1 Monkey kidney fibroblast G5-G9 ND COS-7 Monkey kidney fibroblast G5-G9 ND CHO Chinese hamster ovary G10 ND QS Human synoviocyte G10 ND HepG2 Human liver hepatoblastoma G10 ND Jurkat Human acute T-cell leukemia G7-G10 ND SW480 Human colon adenocarcinoma G10 ND COLO 320D Human colon adenocarcinoma G10 ND SW837 Human rectum adenocarcinoma GIO ND U-937 Human histiocytic lymphoma G7-G9 ND HF1 Primary human fibroblast G10 0.9 x 10-4 RM1 Rat mesothelial G5 2.5 x 10-5 YB2/0 Rat myeloma G7 ND ND, not determined. Downloaded by guest on October 3, 2021 Genetics: Kukowska-Latallo et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4899

Electron Microscope Analysis of DNA/Dendrimer Com- DNA migration was not observed with dendrimers G3 and plexes. DNA/dendrimer complexes were analyzed by electron below (at any charge ratio), suggesting that effective complex microscopy using standard tungsten staining and rotary (450) formation did not occur with these polymers, but was seen with shadowing procedures on a carbon grid. DNA (0.5 jig/ml) was all other generations of both core types of PAMAM dendrim- complexed with either G10 (EDA) or G4 (EDA) dendrimers ers (data not shown). at a DNA-to-dendrimer charge ratio of 1:10 in 1 mM Tris (pH The Ability of DNA/Dendrimer Complexes to Transfect 7.8) and then placed into media in either the presence or Cells. Based on reports describing the transfection of cells absence of DEAE-dextran (0.5 ,uM). using DNA/polylysine complexes formed at positive charge Statistical Analysis. Statistical analyses were performed excess, initial transfection studies using DNA/dendrimer com- using SYSTAT 5.2 software for Macintosh. Errors were calcu- plexes were performed with charge ratios from 1:1 to 1:50. lated as standard deviations and differences between samples G4-G10 Starburst dendrimers of both core types were em- were analyzed by analysis of variance. ployed, because these dendrimers demonstrated the ability to complex DNA. In experiments using Rat2 cell line, a definitive relationship between dendrimer generation and transfection RESULTS efficiency was observed wherein an exponential increase in The Formation of DNA/Starburst Dendrimer Complexes. transfection efficiency was seen with increasing generation When DNA is mixed with PAMAM dendrimers at room from G5 to G10, with a plateau in activity after G8 (Fig. 24). temperature, even for a period as short as 5 min, a charge- This paralleled the increase in surface charge on the den- based complex forms. Analysis of the formation and stability drimer. Other studies using dendrimers of a specific genera- of this DNA/dendrimer complex was performed by examining tion, modified to have either decreased or increased surface charge neutralization ofthe complex as it retards the migration charge, demonstrated decreased and increased transfection of the plasmid DNA during agarose gel electrophoresis. It was efficiency, respectively, due to these modifications (data not found that G7 PAMAM dendrimers of both core types were shown). It was fortuitously observed during these studies that able to fully retard DNA mobility in gels; however, retardation DEAE-dextran placed into buffer containing the DNA/ was complete only when an equivalence or an excess of dendrimer complexes enhanced transfection efficiency (Fig. dendrimer amine groups (as compared with phosphate groups 2B). on the DNA) was present (Fig. LA). At charge ratios where the To evaluate the relative efficiency of dendrimer-mediated dendrimer was in greater than a 5-fold excess, the complex transfection, cells were also transfected with plasmid DNA appeared to have a net positive charge as the DNA migrated using three commercially available cationic lipid preparations. toward the cathode. DNA/dendrimer complexes formed Transfection efficiency using G9 dendrimer in the presence of readily in NaCl concentrations from 50 mM to 1.5 M (Fig. 1B) DEAE-dextran was 20,000- to 40,000-fold more efficient than and in pH from 5.0 to 9.8 (Fig. 1C). Disruption of the complex that achieved with DEAE-dextran (Fig. 2A) and 10- to 100- required strong ionic detergents, such as SDS, and was not fold greater than the transfection achieved with commercially observed under any physiological condition. Retardation of available cationic lipid preparations in six of eight cell lines

A k 1\ +0 4, g\ko,\ .,\ ",, \ \ Q. z 1<0 z IZ) Q3. .<0 Charge ,Ocp 0 rp . rv \. 1". 1.1. jj.. ratio

FIG. 1. Analysis of the formation of DNA/ 1 2 3 4 5 6 7 8 9 10 11 12 dendrimer complexes at various charge ratios and in different buffer conditions. G7 (NH3) dendrimer was complexed with linearized plasmid DNA initially in 10 B mM Tris buffer (pH 7.5) with 100 mM NaCl, at a variety Z~~~ 4z)

CD 120 0o (D 70- o ~~~~~~~~~40- 60- '-80- 50- U) 30- 0. 60- 40- .4 ~~~~~~20 30- C40- o100~ ~ ~ ~ ~ ~ Hor o ~~~~~~~~~~~~~~~~~~~~~~~20- O 20 1010

0 0. 0* 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Hours FIG. 3. The uptake and cellular localization of 32P-labeled plasmid DNA transfected into Rat2 cells using G7 (NH3) dendrimer complexed at 1:10 DNA-to-dendrimer charge ratio. The uptake and distribution of plasmid DNA complexed with dendrimer (-) was compared with the uptake of plasmid DNA alone (U) and plasmid DNA complexed with dendrimer, but incubated with the cells in the presence of 5 mM NaN3 (A). Uptake was determined in whole cells (A), cell nuclear preparations (B), or cell membrane preparations (C). Enhanced uptake is observed with the DNA/dendrimer complex in the whole cell and nuclear fractions at the 3- and 6-h timepoints and is absent after treatment of the cells with NaN3. mM sodium azide, Fig. 3 A-C) or specific inhibitors of concentration of DEAE-dextran (Fig. 4B), suggesting that the endocytosis (deoxyglucose and cytocholasin B; data not dextran alters the nature of the complexes. shown) inhibits the enhanced uptake of DNA/dendrimer complexes. U937 cells demonstrated identical uptake kinetics DISCUSSION with DNA/dendrimer complexes (data not shown), suggesting this activity was not particular to a specific cell type. Our studies indicate that Starburst dendrimers can be used to Analysis of DEAE-Dextran Enhancement of DNA/Den- mediate highly efficient, nonspecific in vitro transfer of genetic drimer Transfection. Given that positively charged dextran material into eukaryotic cells. G3-G10 Starburst PAMAM enhanced transfection in certain cell lines and that cells treated dendrimers with either EDA or NH3 cores form stable com- with dimethyl sulfoxide to permeabilize their membranes did plexes with DNA under most physiologic conditions, and not demonstrate enhanced transfection efficiency with DNA/ G5-G10 dendrimers are capable of mediating transfection. dendrimer complexes (data not shown), it appeared that The restriction of transfection capability to these generations enhancement of transfection with DEAE-dextran might be is probably due to the higher number of surface amino groups due to an alteration of the DNA/dendrimer complex rather and the spherical shape of G5-G10 dendrimers. These char- than the cell membrane. To directly assess the effect of acteristics may allow for simultaneous interaction with both DEAE-dextran on DNA/dendrimer complexes, complexes negatively charged phospholipids on cell membranes and were examined by electron microscopy. This analysis revealed DNA. Efficient transfection of DNA complexed with lipids has that plasmid DNA complexed to a high-generation dendrimer been associated with large cationic substitutions, suggesting a (G10) forms large aggregates hundreds of angstroms in diam- similar phenomenon (11). However, the higher-generation eter (Fig. 4A) that are not observed in either G10 (EDA) dendrimers with the greatest surface charge density form large dendrimer preparations without DNA (data not shown) or aggregates with DNA that appear to be less efficient at DNA/dendrimer complexes formed with a lower-generation mediating transfection in certain cell types. This may happen (G4) dendrimer (Fig. 4C). The large aggregates that formed because the large size of these complexes either prevents with G10 (EDA) dendrimer/DNA complexes were not ob- uptake by the cell, prevents DNA transport to the nucleus once served when these complexes were placed in an appropriate inside the cell, or inhibits the transcription of the DNA.

;0 .4 M5 . C

+1 q _ 5 .,* ;,* X. ;., ,

Plasmid DNA (2.9 kb) was complexed at 1:10 charge ratiowithG (EDA)dendrmer(A)G (EDA)dendrimerinthe * X sjE DEAE-dextran~~~~~~~~~~~~~~~~~~~~~I^ s^ ab g_ ,1m.~|| l* ll ll

.iSv**l2sxF.t8te_iBies l5- 1 FIG. 4. Elecro micogaph o DN/dndrme coplxes. Amagnfictio of X4 0 a sdt xmn h opee br 20n)

FIG. 4. Electron micrographs of DNA/dendrimer complexes. A magnification of X45,000 was used to examine the complexes (bar =220 nm). Plasmid DNA (2.9 kb) was complexed at 1:10 charge ratio with G10 (EDA) dendrimer (A), G10 (EDA) dendrimer in the presence of O.S ,uM DEAE-dextran (B), or G4 (EDA) dendrimer (C). Downloaded by guest on October 3, 2021 4902 Genetics: Kukowska-Latallo et al. Proc. Natl. Acad. Sci. USA 93 (1996) High-efficiency transfection is obtained when these DNA/ variety of cell lines in vitro. The lack of toxicity, high trans- dendrimer aggregates are changed into condensed, toroid fection efficiency, stability of DNA/dendrimer complexes, and DNA particles that appear to maintain an interaction with the the potential to produce this material under Good Manufac- dendrimers. This may be the result of dispersion of the turing Practices conditions suggest that this transfection aggregates in the presence of defined quantities of a molecule method may have utility in in vivo applications. Because with lower (positive) charge density, such as DEAE-dextran. dendrimer-antibody conjugates have already been used ex- However, further studies are necessary to define the exact perimentally in diagnostic applications, in radioimmuno- nature of this interaction. therapy and in the imaging of tumors (15, 18, 19), it is possible Dendrimers offer many advantages for the in vitro transfec- that similar forms of antibody-conjugated dendrimers may be tion of cells. The DNA/dendrimer complexes are very soluble useful in targeted gene delivery in vivo. This could enable some in most aqueous solutions, allowing up to 1.0 mg of DNA per therapeutic uses of gene transfer. ml complexed to 65 mg of dendrimer per ml to remain in solution. DNA/dendrimer complexes are stable for many Funding for this work was provided by R43 CA 68820 and the weeks in solution and appear capable of mediating high- Dendritech Corporation. J.R.B., R.S., and D.A.T. are stockholders in efficiency transfection in a wide variety of cell lines, including Dendritech. primary cells and nonadherent cell lines. There is minimal 1. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) in Molecular cytotoxicity when transfections are performed with DNA/ Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, dendrimer complexes and this appears to facilitate the devel- Plainview, NY), 2nd Ed., pp. 1630-1655. opment of cells that stably incorporate transfected DNA into 2. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., their chromosomes. Seidman, J. G., Smith, J. A. & Struhl, K. (1994) in Current An interesting finding from our work is that several of the Protocols in Molecular Biology (Wiley, New York), pp. 911-943. cell lines evaluated in our studies appeared to have different 3. Chowdhury, J. R., Grossman, M., Gupta, S., Chowdhury, N. R., rate-limiting steps in transfection. Differences in the ability to Baker, J. R. & Wilson, J. M. (1991) Science 254, 1802-1805. transfect cells had been noted with cationic lipid preparations 4. Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A. & Felgner, P. L. (1990) Science 247, 1465-1468. (11, 12), but the basis for these differences was not clear. The 5. Andreason, G. L. & Evans G. A. (1988) BioTechniques 6, precise structure of the dendrimers allows the examination of 650-660. this phenomenon by comparing transfection using various 6. Yang, N. S., Burkholder, J., Roberts,;B., Martinell, B. & McCabe, dendrimers in the presence of different enhancing agents. In D. (1990) Proc. Natl. Acad. Sci. USA 87, 9568-9572. most cells, transfection seemed to be limited by the entry of the 7. Akli, S., Caillaud, C., Vigne, E., Stratford-Perricaudet, L. D., DNA complex into the cell, and efficient transfection therefore Poenaru, L., Perricaudet, M., Kahn, A. & Peschanski, M. R. required the use of dendrimers with high surface charge (1993) Nat. Genet. 3, 224-228. density, possibly to trigger endocytosis. Transfection into some 8. Lehn, P. (1993) Pathol. Biol. 41, 658-662. 9. Graham, F. L. & van der Eb, A. J. (1973) Virology 52, 456-467. other cell lines, COS-1 being an example, was not limited by the 10. Lopata, M. A., Cleveland, D. W. & Sollner-Webb, B. (1984) generation of the dendrimer used (over G5). However, these Nucleic Acids Res. 12, 5707-5717. cells required the addition of a lysosomotropic agent, such as 11. Behr, J. P. (1993) Acc. Chem. Res. 26, 274-278. chloroquine, to achieve the most efficient transfection, indi- 12. Johnson, L. G. (1995) Chest 107, 77S-83S. cating that endosomal localization is a rate-limiting step in 13. Tomalia, D. A. & Durst, H. D. (1993) in Techniques in Current transfection. This suggests that different types of cells might be , ed. Weber, E. (Springer, Berlin), pp. 193-245. specifically transfected with different types of dendrimers 14. Tomalia, D. A., Naylor, A. M. & Goddard, W. A., III (1990) based on the cellular metabolism of the DNA/dendrimer Angew. Chem., Int. Ed. Engl. 29, 138-175. 15. Wu, C., Brechbiel, M. W., Kozak, R. W. & Gansow, 0. A. (1994) complex. Bioorg. Med. Chem. Lett. 4, 449-454. One other technique for gene transfer employing dendrim- 16. Frechet, J. M. J. (1994) Science 263, 1710-1715. ers has been described (27). This technique noted maximal 17. Moore, J. S. (1993) Polymer News 18, 5-10. transfection efficiency using a G6 (NH3) Starburst dendrimer 18. Barth, R. F., Adams, D. M., Soloway, A. H., Alam, F. & Darby, and indicated that transfection greatly diminished when higher M. V. (1994) Bioconjugate Chem. 5, 58-66. generations of dendrimers were used. Transfection efficiency 19. Singh P., Moll, F., III, Lin, S. H., Ferzli, C., Yu, K. S., Koski, R. was only 2- to 4-fold greater than that observed with polylysine, K., Saul, R. G. & Cronin, P. (1994) Clin. Chem. 40, 1845-1849. and thus appeared to be 1000- to 20,000-fold lower than what 20. Kallos, G. J., Tomalia, D. A., Hedstrand, D. M., Lewis, S. & was It was also Zhou, J. (1991) Rapid Commun. Mass Spectrom. 5, 383-386. achieved using the currently described methods. 21. Smith, P. B., Martin, S. J., Hall, M. J. & Tomalia, D. A. (1987) associated with significant cytotoxicity, and no benefit from in Applied PolymerAnalysis and Characterization, ed. Mitchell, J. the addition of chloroquine was observed even in cells that (Hanser, Munich), pp. 357-384. showed increased transfection efficiency in the presence ofthis 22. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) in Molecular compound in our studies. The differences in the results Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press), between the two techniques are difficult to explain. However, 2nd Ed., pp. 142-143. the other study employed commercial dendrimer preparations, 23. Gorman, C. M., Merlino, G. T., Willingham, M. C., Pastan, I. & and our analysis of these preparations has revealed that almost Howard, B. H. (1982) Proc. Natl. Acad. Sci. USA 79, 6777-6781. no intact dendrimers were present. It is therefore possible that 24. MacGregor, G. R. & Caskey, C. T. (1989) Nucleic Acids Res. 17, 2365. the other report was actually demonstrating transfection me- 25. Alam, J. & Cook, J. L. (1990) Anal. Biochem. 188, 245-254. diated by a random mix of polymer fragments and/or fuso- 26. Dignam, J. D., Lebovitz, R. M. & Roeder, R. G. (1983) Nucleic genic . Acids Res. 11, 1475-1489. In conclusion, our studies outline the ability of Starburst 27. Haensler, J. & Szoka, F. C., Jr. (1993) Bioconjugate Chem. 4, dendrimers to mediate high-efficiency transfection in a wide 372-379. Downloaded by guest on October 3, 2021