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A general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. Dendrimer / DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes.
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Anna U. Bielinska et al- Application of membrane-based dendrimer/DNA complexes for solid phase transfection in vitro and in vivo
A general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. Dendrimer / DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes.
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A general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. Dendrimer / DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes.
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Application of membrane-based dendrimer/DNA complexes
for solid phase transfection in vitro and in vivo Anna U. Bielinska', Ann Yen, Huai Liang Wu, Kathleen M. Zahos, Rong Sun', Norman D. Weiner, James R. Baker Jr.'", Blake J. Roessler'* Department of Internal Medicine, University of Michigan Health System, 9220 E. MSRB III 0632, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA 'Center for Biologic Nanotechnology, University of Michigan, 5520 MSRB I, 1150 W. Medical Center Drive, Ann Arbor, MI 48109, USA Department of Internal Medicine, University of Michigan Health System, 1500 Chem 1055, 930 N. University, Ann Arbor, MI 48109, USA Department of Pharmaceutics, College of Pharmacy, 1028 Pharm 1065, 428 Church St., Ann Arbor, MI 48109, USA Department of Internal Medicine, University of Michigan Health System, 5522 MSRB I 0680, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA 'Department of Internal Medicine, University of Michigan Health System, 5520 MSRB I 0680, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA Department of Pharmaceutics, College of Pharmacy, 3010 Pharm 1065, 428 Church St., Ann Arbor, MI 48109, USA "Department of Internal Medicine, University of Michigan Health System, 3918 TC 0380, 1500 E. Medical Center Drive, Ann Arbor, MI 48109, USA Received 7 June 1999; accepted 22 October 1999 Abstract In this study a general description of the use of solid support membranes as the device for DNA delivery mediated by PAMAM dendrimers is presented. In contrast to the other DNA carriers, dendrimer/DNA complexes retain the ability to transfect after drying, which enabled coating or incorporation of complexes into poly(DL-lactide-co-glycolide) or collagen-based bioerodable membranes. These studies provide support for the use of this technology for in vitro and in vivo transfection of skin cells. Expression of luciferase or green #uorescent protein from pCF1-Luc and pEGFP1 plasmids indicated that dendrimer/DNA complexes can mediate transfection after dissociation from the solid support and/or when retained on the surface of the membranes. Modi"cation of the membranes by incorporation of an anionic lipid, phosphatidyl glycerol (PG) at 1}5% concentrations, resulted in more e$cient in situ transfection, particularly with dendrimer/DNA complexes formed at the low charge ratios (1}5). We also report data supporting the feasibility of membrane-based dendrimer/DNA complexes, particularly formed at lower than neutralizing conditions, for topical in vivo delivery of DNA to hairless mouse skin. 2000 Elsevier Science Ltd. All rights reserved. Keywords: In vivo transfection; Dendrimers; Biocompatible membranes; Topical drug delivery 1. Introduction Advances in gene therapy technology have extended the classic concept of drug delivery toward the inclusion of DNA as therapeutic agent [1,2]. Therapeutic genes, antisense oligonucleotides or ribozymes can be delivered to cells by various methods of viral and non-viral catego- ries of delivery systems [3}13]. Cationic PAMAM dendrimers have recently emerged as a novel synthetic carrier for DNA transfer [14}19]. * Correspondence address: Department of Internal Medicine, Center for Biologic Nanotechnology, University of Michigan Health system, 1150 W. Medical Center Drive, 9220E. MSRB III 0632, Ann Arbor, MI 48109, USA. Fax: 001-734-763-4151. E-mail address: roessler@umich.edu (B.J. Roessler) Starburst PAMAM dendrimers are spherical, nano- scopic polyamidoamine polymers with a molecular architecture characterized by the regular dendritic branching and radial symmetry [20,21]. Positive charge density due to the presence of protonized primary amine groups on the surface, enables these molecules to form electrostatic complexes with polyanionic biological mac- romolecules, including various forms of nucleic acids. Dendrimers are highly e$cient for in vitro transfection and appear to be non-cytotoxic in the concentrations relevant for gene transfer [14}16]. Other data suggests that these polymers are not immunogenic or carcino- genic, enhancing their potential as vectors for an in vivo gene transfer system [22]. Topical administration of drugs to epidermal cells usually requires the presence of a carrier which en- ables prolonged contact, enhances skin permeability, and 0142-9612/00/$- see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 9 9 ) 0 0 2 2 9 - X A.B, unpublished observation. Fig. 1. Luciferase expression in COS-1, NIH 3T3 and Rat 2 cells transfected with dendrimer/DNA complexes coated on the surface of PLGA membranes. Luciferase expression is presented as relative light units/g of total protein. Columns represent mean values of three repeats ($SD). No cytotoxic e!ects were observed. extends delivery [23}25]. Biocompatible materials com- posed of polymers such as poly(DL-lactide-co-glycolide) (PLGA) or derivatives of cellulose, collagen, "bronectin, etc. have been tested for their feasibility to serve as the platform for the delivery of traditional pharmaceuticals, including various hormones, nicotine, antihypertensives, etc. [26}43]. We pursued the hypothesis that a similar platform can be used for focal and controlled delivery of DNA to eukaryotic cells in vitro and in vivo using dendrimer/DNA complexes. A unique property of den- drimer/DNA complexes is their ability to retain transfec- tional activity after drying and reconstitution in vitro. This feature enabled us to test their feasibility for solid support-based transfections. We present preliminary studies which have used both surface coating or incorpo- ration of dendrimer/DNA complexes into PGLA or col- lagen-based biocompatible membranes as a possible means to facilitate transfection of dermal cells in vitro and in vivo. 2. Results 2.1. In vitro transfection with dendrimer/DNA complexes coated on the surface of PLGA xlms PLGA polymer membranes were initially used to test the hypothesis that DNA/dendrimer complexes coated onto solid support materials retain the ability to transfect cells in vitro. The pCF1-Luc (encoding "re#y luciferase), pEGFP1(encoding green #uorescent protein), and plas- mid DNA were used to detect and assess e$ciency and frequency of transfection. Dendrimer/DNA complexes were generated at 0.1 mg/ml DNA with G5 EDA, G7 EDA and G9 EDA at the charge ratio 10 and 20. Small, (approximately 10 mm) fragments of the PGLA "lm were incubated with the water-based suspension of DNA/dendrimer complexes. They were then air-dried in sterile conditions. Cultures of the adherent NIH 3T3, COS-1 and Rat2 cells were incubated with PLGA mem- branes coated with DNA/dendrimer complexes. All cell lines expressed luciferase, indicating successful transfec- tion (Fig. 1). A low level of the enzyme expression can be directly attributed to small amount of dendrimer/DNA complexes retained on the membranes (10}30% of total), as prepared with the described method. Cells successfully transfected with pEGFP1, identi"ed using #uorescent microscopy, were found on the entire surface of the culture plates with a frequency of 1}5%(data not shown). These results indicate that dendrimer/DNA complexes can dissociate from PLGA membranes and retain trans- fectional activity. 2.2. In vitro transfection with dendrimer/DNA complexes coated on the surface or incorporated into collagen/xbronectin membranes We then attempted to re"ne the solid-phase trans- fection system by incorporating the DNA/dendrimer complexes both into and onto the surface of collagen membranes. We hypothesized that membranes of col- lagen would be more compatible with a topical mode of application and would facilitate the adherence and growth of keratinocytes. Further, the addition of "bronectin-like peptides to the membranes would also enhance adherence of dendrimer/DNA complexes and cells. DNA/dendrimer complexes generated in various DNA concentrations and dendrimer/DNA charge ratios were coated on the surface of collagen/"bronectin}pep- tide membranes (Experimental Protocol). Analysis of the release of the radioactive DNA indicated immediate dis- sociation of the complexes from collagen membranes, which was not unexpected considering the possibility of only weak electrostatic charge interaction between col- lagen and dendrimer complexes (data not shown). Trans- fection of primary human epithelial keratinocytes (PHEK cells) as well as normal human foreskin "bro- blasts (NHF1 cells) cultured on the surface of the mem- branes coated with pCF1-Luc or pEGFP1 complexed with G5, G7 and G9 EDA dendrimers was detected but was very ine$cient. However, this ine$ciency appeared to be cell type dependent since COS-1 and NIH3T3 cells were transfected up to two orders of magnitude more e$ciently than PHEK cells using the same membranes (Fig. 2). In another series of experiments, we tested the delivery of DNA/dendrimer complexes incorporated into col- lagen/"bronectin membranes. The presence of den- drimers retarded the release of the radioactive-labeled DNA, which did not signi"cantly increase during 72 h incubation. However, intracellular DNA uptake by nor- mal human foreskin "broblast (NHF1 cells) cultured on 878 A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 Fig. 2. Luciferase expression in a variety of cell lines (PHEK, NHF1 COS-1, NIH 3T3) transfected with dendrimer/DNA complexes (at 1, 5 and 10 dendrimer/DNA charge ratios) coated on the surface of collagen/"bronectin membranes. Group A represents complexes formed using G5 EDA dendrimers. Group B represents complexes formed using G7 EDA dendrimers. Group C represents complexes formed using G9 EDA dendrimers. Columns represent mean values of three repeats ($SD). Cell viability was 80 to 100% of non-transfected controls. the surface of the membranes was enhanced and pro- longed in the presence of dendrimer complexes and seemed to be more e$cient at the 10 and 20 than 0.1 or 1 dendrimer/DNA charge ratios (Fig. 3A). The e$ciency of transfection increased as a function of the excess of cationic dendrimer (charge ratios'5) pres- ent in the membrane. The expression of the reporter luciferase gene in most cells peaked at 48 h after initiat- ing the cell cultures on the surface of the membranes (Fig. 3B). Increasing amounts of cell-associated radioac- tivity were observed at 72 h, indicating continuous uptake of dendrimer-complexed DNA (Fig. 3A). Cells cultured on the membranes with naked plasmid DNA did not signi"cantly express the transgene. The visual inspection and assessment of cells viability did not reveal cytotoxicity. However, the overall levels of luciferase ex- pression in NHF1 cells, as compared with Fig. 2, were low possibly because most of the DNA/dendrimer com- plexes incorporated into the collagen membranes was inaccesible for cells growing on the surface of the mem- branes. We then assessed the ability of dendrimer/DNA com- plexes to be released from the membranes following treatment with collagenase. Normal human foreskin "broblasts (NHF1 cells) were seeded on the membranes containing broad range (0.5}20) dendrimer/DNA charge ratio of G7 EDA/pCF1-Luc DNA complexes. When membranes were preincubated with the collagenase, ex- pression of transfected luciferase gene increased 2}3-fold depending on the charge ratio of the dendrimer/ DNA complexes (Fig. 4). This indicates that collagen proteolysis of the membrane leads to greater accessibility of complexes and increases the e$ciency of the in situ transfections. 2.3. Ewect of phosphatidylglycerol on the in situ transfection Di!erent concentrations of the anionic lipid phos- phatidylglycerol (PG) were incorporated into col- lagen/"bronectin membranes in order to increase the anionic surface charge di!erential between membranes and DNA/dendrimer complexes. In contrast to unmodi- "ed collagen membranes, DNA/dendrimer complexes were gradually released from the surface of the PG con- taining membranes over a period of 48 h (data not shown). pCF1-Luc DNA complexed with G5, G7 and G9 EDA dendrimers at dendrimer/DNA charge ratio 1 and 5 was coated onto the surface of collagen membranes containing 1, 5 and 10% phosphatidyl glycerol. In situ transfection of COS-1 cells resulted in the expression of luciferase. Presence of 1 and particularly 5% PG resulted in an e$ciency of transfection of G5 and G7 EDA den- drimer/DNA complexes comparable (50}75%, depend- ing on the charge ratio) to the standard solution-based transfection controls. A further increase of the PG con- centration up to 10% inhibited transfection in particular at the charge ratio 5 (Fig. 5A). A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 879 Fig. 3. DNA uptake (A) and transfection (B) of NHF1 cells cultured on the collagen/"bronectin membranes containing incorporated dendrimer/DNA complexes. Panels A1 and B1 show uptake and expression, respectively, using complexes formed using G5 EDA dendrimers. Panels A2 and B2 show uptake and expression, respectively, using complexes formed using G7 EDA dendrimers. Panels A3 and B3 show uptake and expression, respectively, using complexes formed using G9 EDA dendrimers. Values represent the mean of three repeats, SD does not exceed 15% of total. ()*naked DNA; ()*dendrimer/DNAat charge ratio 0.1; ()*dendrimer/DNAat charge ratio 1; ()*dendrimer/DNAat charge ratio 10; ()*dendrimer/DNAat charge ratio 20. Fig. 4. E!ect of the preincubation with collagenase on the e$ciency of COS cell transfection using G7 EDA dendrimer/DNA complexes incorporated into collagen membranes. Columns represent the mean of three repeats ($SD). Cell viability ranged 80 to 100% of control the control transfections. Extending the charge ratio range of dendrimer/DNA complexes up to 20 did not further enhance transfection (Fig. 5B). As previously observed, the PG containing membranes supported transfection more e!ectively in lower dendrimer/DNAcharge ratios (e.g. 1) However, the lower charge ratios are not e$cient for the transfections in solution or on the surface collagen/"bronectin mem- branes without PG. Naked pCF1-Luc DNA coated on 880 A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 Fig. 5. E!ect of phosphatidyl glycerol (PG) containing collagen membranes on dendrimer/DNA transfection e$ciency in COS-1, NHF1 and primary human keratinocytes (PHEK): (A) transfection e$ciencies in COS-1 were compared between complexes used in solution versus complexes coated onto the surface of collagen membranes containing 1, 5 or 10% (wt%) of PG. Columns represent the mean of three repeats ($SD). N indicates no dendrimers, 1 and 5 indicate dendrimer/DNA charge ratio; (B) NHF-1 cells were transfected with complexes formed using G5, G7 and G9 EDA dendrimers at dendrimer/DNA charge ratios of 1, 10 or 20. Columns represent the mean of three repeats. B1 shows results of experiments using dendrimer/DNA complexes coated on collagen/"bronectin/5% phosphatidyl glycerol (PG) membranes. B2 shows the results obtained using dendrimer/DNA complexes coated on collagen/"bronectin membranes without PG; (C) #uorescent photomicrograph showing GFP expression in PHEK transfected with dendrimer/pCF1GFPcomplexes using G5 EDA dendrimer and a charge ratio of 1. No transfected cells were observed on the membranes coated with naked DNA. Magni"cation 200;. the surface of PG containing membranes was used as a control and did not e$ciently transfect COS-1 or NHF1 cells (Fig. 5A and B). Primary human keratinocytes (PHEK) have proven to be a reasonable in vitro model for skin transfection since it is as di$cult to consistently obtain quanti"able levels A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 881 Fig. 5. (continued). of reporter gene expression in these cells, as in the topical applications in vivo. However, in situ transfection of keratinocytes cultured on collagen}PG membranes with pEGFP1 plasmid complexed with G5 EDA dendrimers resulted in the expression of green #uorescent protein in 15}20% of cells present on the surface of the membranes. Interestingly, to obtain this frequency of transgene ex- pression cells needed to be incubated with 10 pg/ml of recombinant human EGF (Fig. 5C). This result again suggests speci"city of cell biology, not only e$ciency of DNA delivery, as an important factor in the e$cient expression of the transgene. 2.4. Collagen}PG membranes for topical delivery to the hairless mouse skin For in vivo transfection, collagen}PG membranes (PG 5wt%) were coated with 50 to 100 g of pCF1CAT DNA alone or complexed with G5 EDA dendrimers at 0.1, 1 or 10 dendrimer/DNAcharge ratios. After drying, the mem- branes were used for in vivo delivery of the complexed DNA to the denuded skin of hairless mice. At the time of harvest, the collagen membranes had been completely reabsorbed by the host skin and no residual membrane could be detected. Skin biopsies were collected after 24 h, homogenized, and level of expression of chloramphenicol transacetylase (CAT) was determined using CAT-ELISA. Membrane-mediated transfections with uncomplexed plasmid did not result in signi"cant expression of the reporter transgene. CAT expression reaching 50}250 g/mg of skin biopsy homogenates, was obtained with complexes formed at 0.1 G5 EDA/DNA charge ratios and 50 g DNA (DNA concentration dur- ing complex formation: 0.05 mg/ml). However, the skin surface distribution of CAT expression was very uneven and localized (Fig. 6A). This di!erence in the transfection e$ciency/expression may be a result of de- ndrimer/DNA precipitates that are generated when com- plexes are formed in the presence of DNA in concentrations'0.01 mg/ml [50] as well as presence of epidermal cells capable of expressing transfected DNA. Analysis of CAT expression in the mouse skin transfected with collagen-PG membrane-supported dendrimer/ DNA indicate that complexes are most e!ective at the low ((1) charge ratios and increase transgene expression 6}8-fold above uncomplexed DNA level (Fig. 6B). The time course of the expression revealed the transient na- ture of the expression with a peak observed at 24 h. Levels of the transgene expression declined approxim- ately 70% by 48 h post-application (data not shown). Histochemical staining of skin sections showed that CAT activity was not continuously distributed over the entire epidermal surface contacted by the membranes. CAT expression was focal and localized to perifollicular areas suggesting that undi!erentiated keratinocytes and/or dermal "brobalasts were the primary cell types transfec- ted (Fig. 7). 3. Discussion Direct topical transfection of therapeutic DNA into skin cells at the site of disease or as a potential reservoir for the systemic distribution of the transgenic product seems to be an attractive strategy for gene therapy of many human diseases [24,25,39]. However, problems associated with gene delivery in general [2,40] are addi- tionally complicated by the protective structure and function of the skin morphology, which is not easily penetrable by charged macromolecules including DNA. Niemiec et al. has previously reported perifollicular deliv- ery and expression of therapeutic DNA using lipo- some}DNA formulations [41]. In this study we present the preliminary studies on the application of two &deliv- ery devices' for in vivo and in vitro transfection. De- ndrimer/DNA complexes are successfully used for the e$cient transfection of cultured cells [14,18] and have a carrier potential for in vivo applications [42}44]; syn- thetic biocompatible polymers are tested and utilized for topical drug delivery [25,32,33]. Successful transfection of cultured cells with de- ndrimer/DNA complexes coated on the surface of mem- branes of PLGA or collagen/"bronectin indicates that desiccated complexes can dissociate from the membranes and transfect surrounding cells. The e$ciency of this process is a function of both the DNA concentration and dendrimer/DNA charge ratio. PLGA and unmodi"ed 882 A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 Fig. 6. CAT expression in hairless mouse skin following topical deliv- ery of dendrimer/pCF1CAT complexes using PG/collagen/"bronectin membranes: (A) CAT activity was determined in a series of 3 mm punch biopsies obtained from the entire surface of skin occluded by mem- branes coated with 50 g of pCF1CAT DNA alone or complexed with G5 EDA at 0.1 and 10 dendrimer/DNA charge ratios. Numbers (1, 2, 5, 6, 7, 8, 11 and 12) indicate biopsies obtained from individual animals, whilst letter designations (a}e) indicate results obtained from separate biopsies in a single animal. Distribution of CAT expression is not uniform across the surface of the treated skin; (B) the mean value of CAT expression from all skin biopsies obtained in an individual animal were plotted (dots represent individual animals). Mean values from each treatment group (charge ratios; N, 0.1, 1, 10) are indicated by horizontal lines. collagen/"bronection membranes did not allow for the prolonged release of dendrimer/DNA complexes in vitro in the absence of exogenous collagenase. Dendrimer/ DNA complexes also retained a substantial degree of activity when incorporated directly into a matrix of poly- merized collagen. This suggests that prolonged release kinetics and/or controllable rates of release can be achieved when similar membranes are applied to skin and other organ systems for in vivo transfection. Modi"- cation of the membranes by the addition of anionic component (PG) results in an increase in the charge di!erential between dendrimer/DNA complexes and the membrane surface. In vitro results indicate that mem- brane-supported DNA delivery can achieve the levels comparable to solution-based transfection, but at much lower dendrimer/DNA charge ratios. Special considera- tion should be also pay to the dispersity and surface distribution of the dendrimer-complexed DNA. We con- tinue to study the broad range of physico-chemical as well as technological aspects of complex/membrane preparations. Topical application of the dendrimer/DNA containing membranes onto denuded skin of hairless mice resulted in detectable expression of the transgenic CAT when pCF1CAT DNA was delivered as a dendrimer/DNA complex. Naked DNA coated on the surface of the mem- branes did not e$ciently transfect dermal cells. Our data points to at least two dendrimer and DNA-related fac- tors identi"ed as inhibiting e$cient transfection: the pre- cipitation of DNA present in some dendrimer/DNA/ membranes formulation, versus low stability of the naked, non-complexed DNA [45]. In conclusion, we have established the feasibility of a solid-phase dendrimer/DNA/membrane system for the gene transfer both in vitro and in vivo. We believe that our results indicate that transfection is a property speci"c to the assembled membranes and not the individual components per se. Support for our hypothesis was found in the attempts to obtain in vitro transfection using Lipofectamine- or Lipofectin-DNA formulations coated and dried on the surface of PLGA and collagen membranes were unsuccessful (data not shown). De- ndrimer-DNA complex containing membranes can also be desiccated with retention of transfection activity fol- lowing rehydration. This is in contrast to liposomal transfection systems, which do not appear to have consis- tent stability when subjected to desiccation. Further re"nement of the technology will no doubt allow for improvements in the e$ciency of gene transfer and speci- "c targeting of cells within living skin and other organ systems. 4. Experimental protocol 4.1. Dendrimer synthesis The synthesis of Starburst PAMAM dendrimers has been previously described in detail [14,16,29]. Genera- tion (G) 5, 7 and 9 of EDA core dendrimers with molar masses of 28, 826, 116, 493, and 467, 162 Da and numbers of primary surface amine groups of 128, 512, 2048 (re- spectively) were employed in these studies. 4.2. Preparation of membranes PLGA membranes were prepared by dissolving poly(DL-lactide-co-glycolide) (75 : 25 M ` 75 000}120 000, Sigma) polymer in chloroform (10% wt/vol) and pouring the solution onto the surface of sterile siliconized Pyrex A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 883 Fig. 7. In situ histochemical staining for CAT activity in hairless mouse skin treated with membrane bound G5 EPA/pCF1CAT complexes. Sections (5 m) obtained from two animals (panels A and B, as well as C and D represent sections obtained from an individual animal) treated with membrane-bound dendrimer/pCF1CAT complexes were stained for CAT expression as described in Materials and Methods. Representative photomicrographs were then obtained (panels A and C 200;, panels B and D 400;). Arrows indicate perifollicular localization of the brown stain indicating cells transfected with pCF1 CAT. dishes. Chloroform was evaporated under bone dry ni- trogen and the membranes were carefully removed from the glass surface and cut into 4 mm circles using a sterile skin biopsy punch device. PLGA membranes were stored at room temperature until use. Collagen bilayer membranes were made by alkaline- initiated polymerization of a Type-I bovine collagen (Cell Prime, Collagen Biomaterials, Fremont, CA) solution using phosphate-bu!ered saline, pH 7.2 (Life Technolo- gies, Grand Island, NY) as a diluent. The concentration of Type-I collagen in both layers of the bio"lm was 2.2 mg/ml. The base layer of the bio"lm was cast into the bottom of two well chamber slides (Nalge Nunc Interna- tional, Naperville, IL) using a total volume of 1 ml/well. The base layer was polymerized for a period of 24 h at 373C prior to the application of the top layer. The top layer consisted of a total volume of 500 l collagen solu- tion containing 5% phosphatidylglycerol (wt/wt%, Avanti Polar Lipids, Alabaster, AL). Membranes for in vitro transfections were prepared in similar way with the exception that the membranes contained "bronectin- like peptides (1 mg/ml, Sigma). Membranes were allowed to polymerize and cure for a minimum of three days at 373C in a humidi"ed atmosphere prior to the application of the PAMAM dendrimer}DNA complexes. For in vitro studies dendrimer/DNA complexes pre- pared in 25}50 l of water at the indicated charge ratios were coated on the surface of the bilayer, or incorporated into the top layer during membrane polymerization. For in vivo studies membranes were coated with 10}100 g of pCF1CAT DNA alone or complexed with E5 EDA den- drimers at 1 and 5 dendrimer/DNA charge ratios. Den- drimer/DNA complexes were prepared in 100 l of water and incubated for 10 min at room temperature (RT). The complexes were then overlaid on the surface of the mem- branes. Coated membranes were air-dried in a laminar #ow hood for 1}2 h at RT prior to use. 4.3. Plasmids The following reporter plasmids were employed in these studies: pCF1-Luc, pCF1CAT and pEGF1 (Clontech). 884 A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 These plasmids have been described in detail previously [46}48]. Plasmid DNA was ampli"ed in bacteria and then isolated by double cesium chloride gradient [49] to ensure endotoxin-free DNA preparation. 4.4. Preparation of plasmid DNA/dendrimer complexes Dendrimers were diluted to an appropriate con- centration in water and all solutions were stored at 43C until required. DNA/dendrimer complexes were formed by incubating the two components together in 100}200 l of water for a minimum of 10 min at room temperature. Charge ratios of dendrimer to DNA were based on the calculation of the electrostatic charge present on each component, the number of ter- minal NH
groups on the dendrimer versus the number
of phosphate groups in the DNA as previously described [14,15,45,50]. 4.5. Cells and media COS-1, NIH 3T3 and Rat2 cells were maintained in D-MEM medium (Gibco BRL) with 5}10% FCS (Hyclone), 1% penicillin}streptomycin and 2 mM L-glutamine. NHF1 cells were cultivated in RPMI 1640 (Life Technologies) supplemented with 10% fetal calf serum, 1% penicillin}streptomycin, 2 mM L-glutamine, 50 M 2-mercaptoethanol, and 1 mM non-essential amino acids. Primary human epithelial keratinocytes (PHEK) were purchased from Clonetics and grown in keratinocyte SFM medium (Clonetics). For transfection experiments cells were seeded and grown at the subcon- #uent densities 50}70%. All cell lines were incubated at 373C in 5% CO2. 4.6. In vitro transfections Transfections with dendrimer/plasmid DNA com- plexes were performed and analyzed using assays for luciferase activity expression from pCF1-Luc and pEGFP1 reporter plasmids [46,48]. Indicated amounts (g) of pCF1-Luc or pEGFP1 (coding green #uorescent protein) were mixed with dendrimers at a variety of dendrimer to DNA charge ratios (ranging from 1 to 50) in water and were allowed to form complexes for 5 to 10 min at RT. For standard solution-based transfections 24-well plates were seeded 24 h before the transfection with ap- proximately 2;10 cells per well. The dendrimer/DNA complexes were added directly to serum-free medium and transfection occurred for 3 h at 373C. Following incubation with dendrimer/DNA complexes, cells were washed with serum-free medium and returned to com- plete growth media. The cells were harvested 24 or 48 h following transfections and assayed for the expression of luciferase. Cells were seeded directly on the surface of the mem- branes in the presence of medium supplemented with 5% FBS, and incubated for 3}4 h followed by 24}48 or 72 h incubation in the appropriate full growth medium. In all the cases luciferase activity was determined by measuring the light emission from 10 l of cell lysate incubated with 2.35;10\ moles of luciferin substrate (Promega, Technical Bulletin No.101). Light emission was measured in a chemiluminometer (LB96P, EG and G Berthold), and adjusted to the protein concentration of the sample. The protein concentration in the cell lysates was mea- sured in a standard protein assay (DC protein assay, Bio-Rad Richmond, CA). 4.7. Collagenase treatment Collagen membranes with dendrimer/DNA complexes incorporated into the "lm were incubated at 373C with 0.1 mg/ml collagenase (Sigma blend, CC8301). After 30 min incubation, the enzyme solution was removed and "lters continued to incubate by 30 min. Later, ap- proximately 5;10 cells/cm was seeded in the full growth medium, and incubated 24 h before harvesting. 4.8. Animals and in vivo transfections All animal experiments were approved by the Univer- sity Committee on the Use and Care of Laboratory Animals and performed in accordance with ACVM guidelines. Male hairless mice (Skh-hr-1, 60 days old, Charles River Breeding Laboratories, Wilmington, DE) were anesthetized with 30 mg/kg intraperitoneal injec- tion of sodium pentobarbital. The #ank skin of the ani- mals was stripped using cellophane tape a total of 15 times. The membrane was then placed over the stripped area and the edges of the membrane were adhered to the skin using cyanoacrylate glue. The membrane was then covered using an occlusive dressing of petrolatum gauze and sterile gauze wraps to prevent removal of the mem- branes by the animal. The animals were then placed in individual cages for 24 or 48 h at which time they were sacri"ced and the skin processed as described below. 4.9. Harvest of skin for transfection assays At 24 or 48 h the animals were sacri"ced with a lethal dose injection of sodium pentobarbital. The self-adherent wrap was then unwrapped and punch biopsies of skin, either 3 or 4 mm in diameter (Baker Biopsy Punch), were then obtained from the area exposed to treatment. A to- tal of seven to 10 biopsies were typically collected and placed in Eppendorf tubes. Generally, a maximum of four such biopsies were placed in one Eppendorf tube. The punch biopsies were snap frozen and stored at !703C until the extraction procedure was undertaken. A.U. Bielinska et al. / Biomaterials 21 (2000) 877}887 885 4.10. Extraction of protein from skin tissues of hairless mice 100 l of CAT lysis bu!er (CAT ELISA, Boehringer, Mannheim, GmbH) was added to each tube containing the skin tissues and mixed by vortexing for a few seconds. The tubes containing skin tissue were always kept on ice. A probe sonicator (Micro Ultrasonic Cell Disruptor, Kontes Inc.) was used to homogenize the skin in each tube under the following conditions; 40 W and output: 60 W (range from 0}100). The samples were sonicated two times and each time sonication consisted of 7 pulses. The interval between the two sonications was approxi- mately 10 min. The samples were then centrifuged at 15 000 rpm and 43C for 20 min. The supernatants from each tube were then pooled and sonicated a total of six times using the conditions described above. 4.11. CAT ELISA To quantify chloramphenicol acetyltransferase (CAT) expression, 50}100 l of skin homogenate was analyzed in CAT ELISA (Boehringer, Manheim, GmbH). The amount of CAT protein was adjusted to the protein concentration of the samples. 4.12. Histochemical staining for CAT activity Skin punch biopsies (4 mm) were "xed, para"nized and serial sections (5 m) were obtained. Histochemical stain- ing of CAT activity in skin sections was performed using CAT staining kit from Boehringer Mainheim. According to the manufacturer's recommended staining procedure. The Old Hatchett's Brown precipitate is produced by chloramfenicol acetyltransferase (CAT) activity which generates free sulfhydryl group of CoA, and results in the reduction of ferricyanide to ferrocyanide. The ferrocyan- ide complexes with copper ions to form brown precipi- tate. After 12}24 h incubation at RT, slides were rinsed in water and counterstained with hematoxilin and eosin (H-O). Slides were mounted with 100 l of GVA-mount, and photographed with an Olympus BH-2 microscope. 4.13. Statistical analysis Statistical analysis was performed using Systat 5.2 software for Macintosh. Errors were calculated as stan- dard deviations and di!erences between samples were analyzed by ANOVA. References [1] Gewirtz AM, Sokol DL, Ratajczak MZ. 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