Structure/Function Relationships of Polyamidoamine/DNA Dendrimers as Gene Delivery Vehicles CHAD S. BRAUN,1 JOSEPH A. VETRO,1 DONALD A. TOMALIA,2 GARY S. KOE,3 JANET G. KOE,3 C. RUSSELL MIDDAUGH1 1Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Ave., Lawrence, Kansas 66047 2Dendritic Nanotechnologies, Mt. Pleasant, Michigan 48858 3Valentis Inc., Burlingame, California 94010 Received 23 July 2004; revised 16 September 2004; accepted 1 October 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20251 ABSTRACT: PAMAM dendrimers are members of a class of polyamine polymers that demonstrate significant gene delivery ability. In this study, a selection of PAMAM dendrimers, spanning a range of sizes (generations 2, 4, 7, and 9) and transfection efficiencies, are characterized by various biophysical methods to search for structural properties that correlate with transfection. Measurements of colloidal properties (size and zeta potential) as a function of charge ratio reveal that highly transfecting dendrimer/DNA complexes have size/zeta potential values between 4 and 8. Circular dichroism (CD) and FTIR spectroscopy of complexes confirm the DNA component remains in B form when associated with all dendrimer generations up to a 5:1 charge ratio (Æ). Isothermal titration calorimetry and differential scanning calorimetry detect changes that are related to polymer structure and charge ratio but do not directly correlate with transfection efficiency. Despite DNA structural and stability changes detected by CD, FTIR, DSC, and ITC that are similar to those seen with other cationic delivery vehicles [e.g., cationic lipids, peptoids/lipitoids, peptides, polyethyleneimines (PEIs), etc.], clear correlations with transfection activity are not readily apparent. This may be due, at least in part, to the heterogeneity of the complexes. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:423–436, 2005 Keywords: dendrimers; DNA; structure/function analysis; gene delivery INTRODUCTION ferent species formed with transfection ability. In a further extension of this approach, the nature of The mechanism by which nonviral gene delivery the polymer itself can be varied and structure/ vehicles transfect cells remains poorly under- function correlations probed. To this end, we stood.1 This is especially problematic because have systematically studied complexes formed it makes the rational design of more effective between plasmid DNA with various cationic vectors very difficult. One approach to this pro- lipids,2–9 different molecular weight polyethyle- blem is to systematically vary the structure of neimines (PEIs),10 and peptoids with altered side such complexes by altering the ratio of delivery chains11,12 as a function of charge ratios to seek polymer to DNA and attempt to correlate the dif- significant correlations with in vitro transfection efficiency. The result of these efforts has so far been somewhat disappointing. This may reflect the wide differences among the various delivery Correspondence to: C. Russell Middaugh (Telephone: 785- 864-5813; Fax: 785-864-5814; E-mail: [email protected]) vehicles as well as the intrinsic heterogeneity of Journal of Pharmaceutical Sciences, Vol. 94, 423–436 (2005) the complexes so far studied. To obviate some of ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association these difficulties, we have now extended this JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 2, FEBRUARY 2005 423 424 BRAUN ET AL. approach to the study of polyamidoamine The representative PAMAM dendrimers inves- (PAMAM) dendrimer complexes. tigated here (G2, 4, 7, and 9) have well-defined The PAMAM dendrimers are polymers contain- chemistry and molecular weights in contrast to the ing both tertiary amines at branch points as well as cationic component of nonviral complexes formed primary amines at the termini (Fig. 1). PAMAM from cationic lipids, peptoids/lipitoids, and PEI, dendrimers (Fig. 1) are formed by exhaustive providing a system in which transfection efficiency Michael addition of ethylene diamine with methyl varies, but with less complication from hetero- acrylate followed by addition of the resulting ester geneity and variable chemistry.10,12 Therefore, core to an excess of ethylene diamine. This syn- we have conducted an extensive biophysical char- thesis gives a high yield of first-generation acterization of PAMAM dendrimer/DNA com- PAMAM dendrimers, with higher generations plexes (PDDCs) with the goal of establishing arising from repetition of the previous two steps. a correlation between their physical properties Generations up to 10 can be formed before fur- and in vitro transfection efficiency. To this end, we ther assembly is limited by the ‘‘de Gennes have investigated the hydrodynamic size and zeta dense packing’’ phenomenon.13 The dendrimers potential of complexes by dynamic light scat- so prepared have a consistent size, structure, and tering and electrophoretic light scattering. The charge characteristic of their generation. Trans- thermodynamics of the complexation process fection efficiency mediated by PAMAM dendri- and the stability of complexes were examined by mers appears to be dependent on dendrimer isothermal titration calorimetry (ITC) and differ- generation, with larger sizes providing higher ential scanning calorimetry (DSC), respectively. efficiency, and the charge ratio of the complexes, The relative affinities of the DNA/dendrimer in- in which a net positive charge is optimal.14 In teractions were determined by ethidium bromide addition to the simple complexation of DNA with dye displacement. The secondary structure of intact dendrimers, further transfection enhance- the DNA component when complexed to dendr- ment by the use of fractured (hydrolysis degraded) imer was investigated using circular dichroism dendrimers15 or addition of cationic excipients (CD) and Fourier transform infrared (FTIR) such as DEAE–dextran have also been reported.14 spectroscopy. Figure 1. Chemical structure of a generation 1 PAMAM dendrimer. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 2, FEBRUARY 2005 STRUCTURE/ACTIVITY RELATIONSHIP FOR PAMAM DENDRIMER/DNA COMPLEXES 425 These same methods were utilized in the In Vitro Transfection Efficiency characterization of a number of other nonviral The transfection efficiency of dendrimer/DNA gene delivery preparations studied previously in complexes was determined by luciferase expres- our laboratory, as indicated above. The current sion in CHO-K1 cells. The CHO-K1 cells were analysis of the dendrimer/DNA delivery system obtained from the American Type Culture Collec- was compared to these previous studies. tion (Rockville, MD). Cultures were maintained in 2 75 cm flasks at 378C and 5% CO2 and grown in MATERIALS AND METHODS Ham’s F-12 media containing L-glutamine and 10% FBS. Cells were split every 4 days using PAMAM dendrimers of generations 2, 4, 7, and 9 standard procedures, and accutase was employed were a generous gift from Dendritic Nanotechnol- for cell lifting. Plates were prepared by adding ogies (Mt. Pleasant, MI). Plasmid DNA pMB 290 8000 cells (e.g., 16% confluence) to each well of (4.9 kbp) and pMB 401 (5.9 kbp, encoding firefly a 96-well plate and incubating for 18–20 h at 378C luciferase), both >95% supercoiled, were provided and 5% CO2 before transfection. by Valentis, Inc. (Burlingame, CA). The former Approximately 1 mg of PAMAM dendrimer of (pMB 290) was used for all the characterization each generation was dried under vacuum with studies while the latter (pMB 401) was used for silica gel desiccant to remove methyl alcohol until the in vitro transfection studies. The luciferase a stable weight was achieved. The pMB 401 and marker gene was under the control of the CMV 10 mM Tris buffer (pH 7.4) used for reconstitution promoter. Ethidium bromide was purchased from of the dendrimers was filtered through a 0.22-mm Molecular Probes (Eugene, OR). Dulbecco’s mod- filter. Appropriate volumes of polymer and DNA ified Eagle media was acquired from Cambrex were mixed to prepare complexes at a 25 mgmLÀ1 (Rutherford, NJ). Fetal bovine serum was pur- DNA concentration. Complexes at various charge chased from Atlanta Biologicals (Nocross, GA). ratios were diluted to a final transfection DNA Accutase was obtained from Innovative Cell concentration of 2 mgmLÀ1 with Opti-MEM. Technologies (San Diego, CA). Opti-Mem was Immediately before transfection, the cells were acquired from Invitrogen (Carlsband, CA). 1,2- washed once with PBS followed by addition of Dioleoyl-3-trimethylammonium propane (DOTAP) 100 mL of solution containing complexes (200 ng of was obtained from Avanti Polar Lipids (Alabaster, pMB 401) to each well. Cells were incubated with AL). Tris and phosphate buffer salts were pur- complexes for 5 h. The 100 mL of transfection solu- chased from Sigma (St. Louis, MO). Nano-purified tion was then removed and replaced by 100 mLof water was used for all buffer preparation. culture media. The cells were incubated for a fur- ther 48 h at 378C and 5% CO2. Preparation of Complexes Luciferase expression was assayed using the Luciferase Assay SystemTM from Promega The dendritic polymers were received at high (Madison, WI) following the supplied protocol. The concentration (>10% w/w) in methyl alcohol, and cells were lysed with Reporter Lysis Buffer for were diluted directly into buffer for analysis 30 min. Expression was analyzed in 96-well plates unless otherwise stated. The plasmids were with a FluostarTM Galaxy microtiter plate reader diluted from a stock solution. The concentration (BMG, Offenburg, Germany). Automated addition of DNA solutions was determined using their UV of substrate buffer was employed and the lumines- absorbance at 260 nm (A ) and a molar extinc- 260 cence was then measured. The amount of protein tion coefficient of 0.02 (mgÀ1 cmÀ1 mL). Individual in each well was determined by Coomassie Blue dilutions were performed for DNA and dendrimer assay (Promega). The data were normalized to the at each charge ratio, calculated as the mol of total protein content and reported as relative light dendrimer primary amines per DNA phospha- units per milligram of protein (n ¼ 3). tes.