BioconJugate Chem. 1093, 4, 372-379
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Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture+ Jean Haensler and Francis C. Szoka, Jr.’ School of Pharmacy, University of California, San Francisco, California 94143-0446. Received May 7, 1993”
Cascade polymers also known as Starburst dendrimers are spheroidal polycations that can be synthesized with a well-defined diameter and a precise number of terminal amines per dendrimer. We show, using luciferase and beta-galactosidase containing plasmids, that dendrimers mediate high efficiency transfection of a variety of suspension and adherent cultured mammalian cells. Dendrimer-mediated transfection is a function both of the dendrimer/DNA ratio and the diameter of the dendrimer. Maximal transfection of luciferase are obtained using a diameter of 68 A and a dendrimer to DNA charge ratio of 611 (terminal amine to phosphate). Expression is unaffected by lysomotrophic agents such as chloroquine and only modestly affected (2-fold decrease) by the presence of 10% serum in the medium. Cell viability, as assessed by dye reduction assays, decreases by only 30% at 150 pg dendrimer/mL in the absence of DNA and about 75 % in the presence of DNA. Under similar conditions polylysine causes a complete loss of viability. Gene expression decreased by 3 orders of magnitude when the charge ratio is reduced to 1:1. When GALA, a water soluble, membrane-destabilizing peptide, is covalently attached to the dendrimer via a disulfide linkage, transfection efficiency of the 1:l complex is increased by 2-3 orders of magnitude. The high transfection efficiency of the dendrimers may not only be due to their diameter and shape but may also be caused by the pK,)s (3.9 and 6.9) of the amines in the polymer. The low pK:s permit the dendrimer to buffer the pH change in the endosomal compartment. The characteristics of precise control of structure, favorable pKa’s, and low toxicity make the dendrimers suitable for gene-transfer vehicles.
INTRODUCTION Molecular biologists have identified the chromosomal defects in a large number of human hereditary diseases, raising the prospects for cures using gene therapy. This emerging branch of medicine aims to correct genetic defects by transferring cloned and functionally active genes into the afflicted cells. At present, viruses are the most efficient vectors for gene transfer (1, 2 ) , but potential risks associated with viruses have catalyzed a search for synthetic DNA-delivery systems (3). Early work showed that polycations such as polylysine and DEAE-dextran promote the uptake of proteins (4)and single- and doublestranded polynucleotides into animal cells (5,6),and since then, polylysine-based vectors for gene transfer have been extensively studied (7). However, the linear polycations are relatively cytotoxic and by themselves not very efficient, which limits their usefulness for transfection of cella in culture. In spite of these drawbacks, polylysines have a number of advantages: they help to assemble DNA into a compact structure, destabilize cell membranes, and provide a handle for the attachment of other effectors to the nucleic acid. Neutralization and condensation of DNA by polylysines into small (ca. 100 nm) toroid-like structures promotes
* Address correspondence to this author.
+ Abbreviations
used: DME, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid;FCS, fetal calf serum;HBS, Hepes buffered saline (10mM Hepes; 150mM NaC1, pH 7.3); Hepes, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid); MEM, minimal essential Eagle’s medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Tris, tris(hydroxymethy1)aminomethane; PAMAM SD54 and SD68, polyamidoamine Starburst dendrimer 54 and 68 A in diameter; pLysll5, poly(L-lysine) with an average chain lengh of 115 monomers. e Abstract published in Advance ACS Abstracts, September 1, 1993. 1043-1802/93/2904-0372$04.Q0l0
the endocytosis of the nucleic acid into cells in vitro (8). The endocytic process can be further stimulated by the covalent attachment to the polycation of specific ligands like transferrin ( 9 ) ,asialoorosomucoid (IO),or insulin (11). When polycation transfection procedures are based upon receptor-mediated or fluid-phase endocytosis, a large fraction of the endocytosed DNA becomes trapped in intracellular vesicles and is ultimately degraded in the lysosomes. Lysosomal degradation can be partially bypassed by the addition of lysomotrophic agents such as chloroquine during the transfection (12) or by the attachment of endosome disrupting agents to the polylysine, such as inactivated viruses (13)or viral fusogenic peptides (14). The ability of polylysine-DNA complexes to transfect cells is strongly dependent upon the presence of these effectors. As an alternative to polylysine for transfection, we describe a nonlinear polycationic cascade polymer which combines the DNA-binding and delivery properties of polylysine and the lysomotrophic effects of weak bases (15). We show that polyamidoamine (PAMAM) cascade polymers, a well-defined class of dendritic polymers synthesized from methyl acrylate and ethylenediamine (161, are well-tolerated by cells and when complexed to plasmids encoding reporter genes mediate highly efficient transfection of a wide variety of cells in culture. In addition, the covalent attachment of the amphipathic peptide GALA (17) to the cascade polymer can significantly enhance transfection efficiency in both primary cells and cell lines. EXPERIMENTAL PROCEDURES Materials. PAMAM cascade polymers synthesized from an ammonia initiator core (generation 2-10) were obtained from Polysciences,Inc.(Warrington,PA) and are designated as Starburst dendrimers. When needed, dendrimer solutions were concentrated using a Savant 0 1993 American Chemical Society
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SCllO Sped Vac system. Similar results to those reported here are obtained when the cascade polymer was synthesized in our laboratory using the published method (16) but starting from tris(2-aminoethy1)amine (unpublished results, C. Redemann, J. Haensler, and F. Szoka). The commercially available dendrimers should be analyzed on a calibrated gel permeation column to insure that the material conforms to the specified diameter, since we have received a few lots that did not conform to specifications. Poly(L-lysine)hydrobromide with an average chain length of 115 lysine residues (pLysll5) was obtained from Sigma Chemical Co. (St. Louis, MO). N-Succinimidyl 342pyridy1dithio)propionate (SPDP) was obtained from Pierce (Rockford, IL). GALAcys (WEAALAEALAEALAEHLAEALAEALEACAA) a cysteine-containing analog of the amphipathic peptide GALA was synthesized at the UCSF Bioresources Center, purified, and analyzed essentially as previously described for GALA (17). Modification of SD54 with GALAcys. The PAMAM dendrimer of the fifth generation (54 A in diameter, SD54) was modified following the scheme in Figure 1. Functionalization of SD54 with SPDP. The dendrimer (66 pmol terminal amines, 15 mg) in 0.5 mL of water was diluted with 0.75 mL of 0.1 M phosphate buffer (pH 8.01, and 0.75 mL of a 15 mM solution of SPDP in ethanol was added dropwise. The reaction mixture was stirred for 1 h under argon and fractionated on a Biogel P2 column (2.8 X 20 cm) eluted with 0.1Mphosphate buffer (pH 7.4). The fractions containing 3-(2-pyridyldithio)propionatemodified dendrimers (PDP-SD54 with an average of 16 dithiopyridine groups per particle) were pooled together and concentrated to a final volume of 3 mL. Reaction of PDP-SD54 with GALAcys. One milliliter of a 10 mM solution of GALAcys in a 0.1 M phosphate
buffer (pH 7.2) was added dropwise to 1 mL of the concentrated PDP-SD54 solution. The mixture was stirred overnight under argon, then the GALA conjugate was purified by fractionating the reaction mixture on a calibrated Sephade G75-120 column (2 X 90 cm) [calibration kit: Sigma MW-GF-70 containing aprotinin (6500), cytochrome C (12 4001, carbonic anhydrase (29 OOO), albumin (66 OOO), and blue dextran (2 OOO OOO)]. The column was eluted with 0.1M phosphate buffer (pH 7.4). The fractions containing the conjugate, which eluted with an apparent molecular weight of about 50 OOO, were pooled, concentrated, and dialyzed against HBS (10 mM Hepes; 150mM NaC1, pH 7.3). The dialyzed material was diluted with HBS to 1 mg of dendrimer/mL and sterile filtered through a 0.45-pm Millipore membrane. Expression Vectors. The plasmids pCLUC4 (18) encoding firefly luciferase and pCMV-BGal(19) encoding @-galactosidasewere generous gifts from Dr. M. Cotten (Institute of Molecular Pathology, Vienna, Austria) and Dr. G. McGregor (Howard Hughes Medical Institute, Houston, TX), respectively. Plasmids were grown in Escherichia coli, extracted by the alkali lysis technique, and purified by centrifugation in equilibrum CsCl gradients. The purity of the plasmids was checked by electrophoresis on a 0.8% agarose gel and the DNA concentration was determined by absorbance at 260 nm. Preparation of Complex. A typical complex was made by diluting 6 pg of plasmid DNA into 330 pL of HBS in a polystyrene tube. The polycation and/or its GALAfunctionalized derivative (2-160 pg) were diluted in 170 pL of HBS and added dropwise to the DNA. When the addition was completed the solution was gently mixed. The formation of the polycation-DNA complex was shown by a gel retardation assay; samples (30 pL) were electro-
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phoresed through a 0.8% agarose gel using a Tris-acetateEDTA buffer system (pH 8.0) and DNA was visualized using ethidium bromide staining. Cells a n d Transfection Protocol. The adherent cell lines CV-1 (monkey fibroblast), HeLa (human carcinoma), and HepG2 (human hepatoma) were provided by the UCSF cell culture facility. Cells were plated at a density of about 5 X lo5 cells per 60 mm culture dish (Falcon) in 3 mL of DME-H21 containing 10% FCS and antibiotics (100unita/mL penicillin, and 100unita/mL Streptomycin). The cells were grown to half confluency at 37 "C in a humidified atmosphere containing 5% C02. In a typical experiment, cells were transfected in 1.5 mL of medium without serum by addition of 0.5 mL of HBS containing 6 pg of plasmid complexed with the indicated amount of polycation. Five hours later the medium was removed and replaced by fresh medium containing 10% FCS. The cells were cultured for an additional 24- or 48-h period and tested for reporter gene expression. The suspension cell lines K-562 (human erythroleukemia),EL-4 (mouse lymphoma), and Jurkat (human T-cells) were obtained from the UCSF cell culture facility and grown in RPMI 1640containing 10% FCS and antibiotics. For the transfection experiment, 1.2 X lo6cells in 1.5 mL of RPMI 1640 containing 7.5 % FCS were introduced into each well of a 6-well plate or rotated in polypropylene tubes and transfected with 6 pg of DNA as described above. Five hours later the cells were transferred to fresh medium containing 10% FCS. This was accomplished after centrifugation of the plate or tubes (1200 rpm for 5 min) and by removing 90% of the transfection medium and replacing it with fresh prewarmed medium. The cella were cultured for an additional 24- or 48-h period and tested for reporter gene expression. Freshly isolated rat hepatocytes were obtained from Dr. M. Bissel (Liver center, UCSF) and plated at a density of 2 X 106 cells per 60-mm culture dish in 3 mL of a medium (hepatocyte medium) composed of 75 % MEM and 25 ?& Waymouths and containing 10% FCS, insulin (lOpg/mL), transferrin (10 pg/mL), dexamethasone (1 pM), and antibiotics (100 units/mL penicillin, 100 pg/mL streptomycin, and 25 pg/mL gentamycin). Cells were grown at 37 OC in a humidified atmosphere containing 5% COz. The cells were transfected 5-6 h later as described above with 6 pg of plasmid in 2 mL of the hepatocyte medium containing 2 % FCS. After an overnight incubation period, the transfection medium was removed and replaced with 3 mL of fresh hepatocyte medium containing 2% FCS. The cells were further cultured for 24 h and tested for reporter gene expression. j3-Galactosidase gene expression was detected 24 h after transfection by histochemical staining of the cells using X-Gal (20). Luciferase reporter gene expression was quantitated 48 h after transfection on cell lysates by measuring the light emission with a bioluminometer (Analytical bioluminescence, San Diego, CA) in the presence of luciferin and ATP (21). The effect of chloroquine on transfection efficiency was studied by performing the transfection in medium containing 100 pM of this lysomotrophic agent. When hepatocytes were transfected in the presence of chloroquine, the transfection step was reduced to 5 h to avoid acute cytotoxicity. Toxicity Assay. The effects of the cascade polymers on cell growth were assessed by measuring total protein content after transfection as well as by using a colorimetric dye reduction assay (22). The effect of the sixth generation dendrimer (68 A in diameter, SD68) was compared to the
effect of polylysine (pLysll5). The polycationswere added to the cells with or without plasmid DNA a t a ratio of 10 terminal amines of the polycation per nucleotide. Cells were plated at a density of about 5 X lo4cells per well in 300 pL of DME H-21 in 96-well trays. After an overnight culture at 37 "C in a 5% C02 humidified atmosphere, the cells were incubated in triplicates with 200 pL of serumfree medium containing 0-60 pg of pLysll5 or SD68. After 5 h the media were replaced with 200 pL of fresh DME H-21 containing 10% FCS, and the cells were cultured for an additional 48 h. Then, 10pL of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazoliumbromide (MTT, 5 mg/mL) was added per well and allowed to react for 2 h at 37 O C . Solubilizing solution (0.4 N HC1 in 2-propanol, 200 pL) was added and the plate incubated for 30 min a t room temperature. Absorbance a t 570 nm was measured using an automatic ELISA plate reader (MR 700, Dynatech Laboratories Inc.) and corrected for background absorbance obtained on cells treated with 6M guanidinium hydrochloride (100% death). Results are expressed as percent reduction in cell viability = {l- [OD570(treated cells) - background]/ [ODsduntreated cells) - background]) X 100. RESULTS
Synthesis and Characterization of Modified Cascade Polymers. One goal of this work was to use the dendrimer as a platform to attach functional groups to polynucleotides such as DNA to create gene delivery vehicles. The fifth generation PAMAM dendrimer was linked to GALAcys, a cysteine-containing analog of the amphipathic peptide GALA, by using standard SPDP coupling chemistry (23)(Figure 1). The fifth generation dendrimer has a hydrodynamic radius of 54 A, a molecular weight of 21 563 and contains 96 terminal amines. The dendrimer was successively functionalized with the heterobifunctional reagent SPDP and reacted with an excess of GALAcys. The resulting conjugate eluted from a calibrated Sephade G75-120 column with an apparent molecular weight of about 50 000 Da. This suggesta the presence of about 10 GALA residues per dendrimer. An average of 13GALA residues were found to be conjugated to one dendrimer when GALA was quantified by using a molar extinction coefficientfor tryptophan of €2= 5570 M-' cm-l (pH 7.5). Since GALA contains eight negative charges per peptide, most of the unmodified amines on the conjugates are probably neutralized at pH 7.4, a supposition that is supported by the weak binding of the GALA conjugate to DNA (vide infra). Binding of Cascade Polymers to DNA. PAMAM dendrimers bind to DNA as demonstrated by retention of the complex at the point of application on an agarose electrophoresis gel (Figure 2). Both the polylysine (lanes 2 and 3) and the cascade polymer (lanes 4 and 5) were able to retard and immobilize the DNA on the gel. Gel retardation is a result of electrostatic and steric effects and suggests the formation of a charge complex between the positively-charged dendrimers and the anionic DNA. Because dendrimer terminal amines have lower pKa than lysine tNH2 (see Discussion), the dendrimer was slightly less effective in inducing gel retardation than polylysine (Figure 2). With polylysine,total retention of the plasmid was observed at a 1:l eNH2 to nucleotide ratio (lane 3); with the dendrimer total retention occurred a t about 1.5:l terminal NHz to nucleotide ratio (lane 5). The exact ratio of total retention varied by one dilution factor among experiments. The GALA-dendrimer conjugate did not immobilize the DNA and affected only slightly the
DNA Transfection Using Cascade Polymers
1
2
3
4
5
6
Bioconlugate Chem., Vol. 4, No. 5, 1993 375
7
8
Figure 2. Effect of pLysll5, SD68, SD54, and GALA-SD54 on the electrophoretic mobility of plasmid DNA. Samples (30 pL) of polycation-pCMV-BGal complexeswere incubated for 20 min and electrophoresed through a 0.8% agarose gel using a Trisacetate-EDTA buffer system (pH 8.0). The complexes were formed as described in Experimental Procedures by mixing 6 pg of pCMV-@Galplasmid diluted in 330 p L of HBS with the following agents in 170 pL of HBS: lane 1,pCMV-PGal alone; lane 2 , 2 pg of pLysll5; lane 3 , 4 pg of pLysll5; lane 4 , 4 pg of SD68; lane 5,6 pg of SD68; lane 6,4 pg of SD54 provided by the GALA-SD54 conjugate; lane 7,160 pg of SD54 provided by the GALA-SD54 conjugate; lane 8, 4 pg of SD54 provided by an equimolar mixture of SD54 and GALA-SD54. After the electrophoresis was completed the gel was stained with ethidium bromide to visualize DNA.
migration of the plasmid even when used in large excess over the DNA (lanes 6 and 7). A combination of GALAdendrimer conjugate and unmodified dendrimer (1:lratio, as used in the transfection assays) partially retained the plasmid (lane 8). Optimization of the Complex for Transfection.An unexpected finding of this work is that PAMAM cascade polymers alone could transfect DNA (e.g. plasmids encoding reporter genes) into cells in culture (Table I; Figures 3-5). The transfection activity was particularly high when an excess of terminal amines to nucleotide was used. To define the parameters controlling gene delivery and expressionby the dendrimers, CV-1 cells were transfected with pCLUC4-dendrimer complexes and luciferase expression measured as a function of the diameter and amount of the dendrimer in the complex (TableI). Transfection was sensitiveto both factors. High luciferase expression required an excess of polycation; a t a low dendrimer input ([dendrimer primary amines] 5 [nucleotides]) cells were only poorly transfected by the complexes. In the concentration range studied, large diameter dendrimers (4 1 40 nm), mediated better transfection efficiencies than the smaller ones. Luciferase expression increased by 2-3 orders of magnitude when the diameter of the complex-forming dendrimer was increased from 40 (generation 4) to 54 A (generation 5). This can be best appreciated from examining the data on a three-dimensionalplot (Figure3). Maximal levels of transfection were obtained (11010light units (LU)/mg cell protein) with the dendrimer of the sixth generation. The sixth generation dendrimer, designated SD68, has a hydrodynamic radius of 68 A and a molecular weight of 43 451 and contains 192 terminal amines. Using SD68 a t a ratio of six primary amines per nucleotide, luciferase expression in CV-1 cells was about a 1000-fold greater than that obtained with an equivalent amount of polylysine 115 (Table I) and a 100fold greater than that obtained with the cationic lipid DOTMA (24,25). The optimized conditions were tested in 10 separate experiments in CV-1 cells; luciferase activities between 2 X lo9 and 3 X 1O1O LU/mg of cell
proteins were obtained. When CV-1cells were transfected with dendrimer in medium containing 10% FCS, the luciferase expression was decreased by about 2-fold, whereas expression decreased by 50-fold in the case of DOTMA (25). A dose response of luciferase activity versus DNA input a t a constant terminal amines/nucleotide ratio of 6/1 was constructed using SD-68 (Figure 4). When the complex was first formed and then diluted to the stated amount of pCLUC4 plasmid, a linear decrease in expression of luciferase was observed (Figure 4). If non-luciferasecontaining plasmid was used to dillute the luciferase plasmid and the dendrimer added, the transfecting activity also decreased in a linear fashion. When the plasmid was first diluted and a constant amount of dendrimer was added (in this case dendrimer/plasmidratio increaseswith the dilution of the pCLUC4 plasmid),transfection activity decreased to a greater extent (Figure 4). Transfection of MammalianCellswith SD68-DNA Complexes at Low Terminal Amines/Nucleotide Ratios. Every cell type examined in this study could be transfected under conditions of excess terminal amines over nucleotides, with PAMAM dendrimer-luciferase plasmid. The cells examined included adherent and suspensioncells and both primary cultures and established lines (Figure5A). Transfectionefficiency of the optimized complexwas also examined using the pCMV-pGal plasmid as the reporter gene. 8-Galactosidaseactivity was detected by a histochemical stain 24 h after transfection and transfected cells counted. The percentage of transfected cells in the population detected by the histochemical stain is indicated at the end of the bar on the graph (Figure 5A). The cell types studied differed in their ability to undergo transfection (up to 80% transfection in CV-1, less than 1% transfection in EL-4 and Jurkat). This variability is a general property shared by all gene delivery systems; the reason for such variable transfection among cell types is not understood yet. At the lower dendrimer input, the transfection efficiency of the dendrimer-DNA complexes was dramatically reduced (Figure 5A,B). Reduced transfection activity with lower terminal amines/nucleotideratios is similarto results seen with polylysines. Thus we examined whether or not treatments that can increase transfection mediated by linear polycations could increase the dendrimer-mediated transfection. Interestingly, transfection was essentially insensitive to chloroquine (not shown) but increased significatively when the fusogenic peptide GALA was attached to the dendrimer. Enhancement of Transfection by Covalent Attachment of the Amphipathic Peptide GALAcys to the Cascade Polymer. The endosome disruptive effects of inactivated viral particles and of viral fusogenic peptides have been exploited to trigger or enhance polylysinemediated gene transfer (13,14).The 30 aminoacid peptide GALA was designed to destabilize lipid bilayers in a pHsensitive manner to mimic the properties of viral fusogenic proteins (17). We attached GALA to the dendrimer to learn if it could increase transfection as do the viral particles or the viral fusogenic peptides. A t a low terminal amines to nucleotide ratio, the transfection efficiency of dendrimer-DNA complexeswas low; however, transfection was significantly improved when 50% of the dendrimer in the complex was replaced for its GALA conjugate (Figure 5B). Under these conditions the conjugate can function a t a low dendrimer/plasmid ratio (about 50:l in these experiments). In the case of some cell types, such as K562, the GALA conjugate was as effective as using the 6-fold
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Table I. Influence of the Amount and Size of PAMAM Cascade Polymer Complexed to pCLUC4 Plasmid on Luciferase Reporter Gene Expression in CV-1Cells generation
diameter,
luciferase activity” 3 6 10 16 25 Starburst Dendrimer 2 22 (8.9 f 0.8)1@ (1.6 f 0.01)105 (1.9 f 0.2)105 (4.1 f 0.06)105 (3.7 f 0.7)105 (4.3 f 0.4)105 (5.7 f 0.9)1@ (7.5 f 0.1)103 (7.7 f 4.5)104 (1.0 f 0.3)105 (3.9 f 0.3)105 (7.5 f 0.05)105 (1.5 f 0.5)106 (1.1f 0.08)106 3 31 4 40 (2.7 f 0.2)103 (2.9 f 0.2)1@ (2.7 f 1.8)105 (9.8 f 4.0)105 (1.2 f 0.3)106 (3.4 f 0.4)106 (7.7 f 1.1)106 5 54 (3.1 f 1.8)104 (6.1 f 0.5)105 (1.1f 0.03)108 (1.5 f 0.2)108 (2.6 f 0.3)108 (6.1 f 0.4)108 (5.9 f 1.1)108 [88%1 [74%1 172% 1 192% 1 (7.5 f 5.8)104 (2.7 f 0.05)105 (4.0 f 0.5)108 (1.0 f 0.1)1O1O (1.3 f O.4)1O1O (1.2 f 0.3)109 (4.8 h 0.61108 6 68 156% 1 153% 1 145%1 [64%1 (1.9 f 0.3)104 (4.8 f 0.1)105 (4.9 f 0.5)107 (2.3 f 0.5)108 (5.1 f 0.7)108 (2.5 f 0.4)108 (7.3 f 0.6)107 7 84 156% 1 143% 1 [26%1 193% 1 E60 % 1 (1.9 f 0.4)104 (5.7 f 0.6)105 (2.8 f 0.9)107 (1.8 f 0.5)107 (3.5 f 0.3)107 (7.4 f 2.0)107 (1.1f 0.3)108 8 95 [60%1 [38%1 [25%1 [90%1 [82%1 177%1 9 107 (3.2 f 2.6)1@ (7.7 f l.2)104 (3.9 f 0.3)107 (1.9 f 0.2)108 (1.9 f 0.4)108 (1.8 f 0.06)108 (1.7 f 0.03)108 [68%1 161% 1 [44%1 [90%1 10 124 (2.4 f 0.3)103 (2.8 f 0.2)103 (7.7 f 0.6)106 (1.4 f 0.07)107 (8.4 f 0.6)106 (1.0 f 0.03)107 (4.4 f 0.02)106 [76%1 [65%1 [60%1 191%Ic PLYS115 (1.6 f 0.5)104 (1.7 f 0.6)105 (1.9 f 0.1)106 (7.3 f 2.3)106 (1.7 f 0.2)107 (1.9 f 0.6)107 IO%]” [88%Ic 157%1 137% 1 [ll%I Luciferase activity in the transfected cells (light units per mg of cell protein) is shown as the mean f range of duplicates. Primary amines/nucleotides. When the transfection txocedure was toxic, the percentage recovery of cell protein in transfected cells compared to nontransfected cells is indicated in brackets.
A
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1
1010
109 108
107 106
105 LL
104
s T
103
U
c
1031 0
-
0
1
T
2
3
4
AMOUNT (pg) OF pCLUC4
Figure 3. Effect of dendrimer diameter and amount in mediating transfection in CV-1 cells (data from Table I).
excess of dendrimer for transfection (Figure 5A,B). These results indicate that GALA is a transfection-enhancing agent probably by catalyzing endosome leakage. At a dendrimer input where transfection was maximal, expression was not enhanced further by GALA. This may be because dendrimers display lysosomotrophic effects (see Discussion). Comparison of Cytotoxicity of PAMAM Cascade Polymer to pLys 115. One index of the toxicity of the transfection procedure is the amount of cell protein obtained from the cultures following transfection. Treatments that resulted in a decrease in the yield of cell protein when compared to nontreated cells are indicated in brackets in Table I. Dendrimer-DNA-induced cytotoxicity seemed to be controlled by three main parameters: the diameter of the dendrimer, the amount added, and whether or not DNA was present. The last factor can be better appreciated when the toxicity of the dendrimer SD68was compared to polylysine 115 on CV-1 cells in the presence and the absence of plasmid DNA (Figure 6). The overall cytotoxicityof SD68
5
6
IN THE COMPLEX
Figure 4. Dose-response of luciferase activity versus the amount of pCLUC4 plasmid used for the transfection. CV-1cells (500 000 cells per 60-mm dish) were transfected in duplicate with increasing amounts of pCLUC4 (0.1-6 pg) complexed to SD68. The complexes were formed as described in Experimental Procedures by adding the dendrimer dissolvedin 170 pL of HBS to the DNA in 330 pL of HBS; 0,complexes were first formed by adding 25 pg of SD68 on 6 pg of pCLUC4 and then diluted in HBS to the stated amount of DNA; 0,the plasmid was first diluted in HBS and 25 pg of SD68 was added; A, a non-luciferase-containing plasmid was added to the diluted pCLUC4 plasmid to keep the total amount of DNA constant a t 6 pg/330 pL and then 25 pg of SD68 was added. Luciferase expression was measured 48 h posttransfection as described in Table I. Each value is the mean & range of duplicate determinations.
was low when compared to that of pLysll5. In the absence of DNA, the LD50 of pLysll5 for CV-1 cells was 25 pg/mL and the LD50 of SD68 was greater than 300 pg/mL. In the presence of plasmid DNA (ratio of 1O:l primary amines of polycation to nucleotide), the cytotoxicity of pLysll5 was not affected while that of SD68 was increased (LD50 of SD68-DNA = 100 pg/mL) but still was significantly less than that of pLysll5. Under the optimized transfection conditions, the concentration of SD68 was 12.5 pg/mL, a level of dendrimer that induced about a 35 % decrease in dye reduction (Figure
DNA
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CELL TYPE 30-80%
A
120
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