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Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers
Reviews in Molecular Biotechnology 90 Ž2002. 339᎐347
Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers Jorg ¨ DennigU , Emma Duncan QIAGEN GmbH, Max-Volmer Straße 4, 40724 Hilden, Germany
Abstract The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency andror restricted applicability. Activated polyamidoamine ŽPAMAM.-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Polyamidoamine dendrimers; Transfection
1. Introduction Gene transfer into eukaryotic cells Žtransfection. is a useful tool for molecular biotechnology. Over the last 35 years a number of different transfection technologies have been developed. Classical technologies include the use of infectious viral vectors, direct introduction of nucleic acids by electroporation or microinjection, and the use of chemicals to create DNA complexes
U
Corresponding author. Fax: q49-2103-29-26-326. E-mail address: [email protected] ŽJ. Dennig..
that attach to the cell surface and are taken up by endocytosis. Each of these approaches has associated disadvantages, the most problematic being cytotoxicity and low transfection efficiency. Activated polyamidoamine ŽPAMAM.-dendrimers provide a new transfection technology that offers significant advantages over classical transfection methods as well as cationic liposomes. Transfection reagents based on this technology provide high transfection efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. In this review we first give a brief overview of transfection and existing transfection technologies, and then discuss the
1389-0352r02r$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 5 2 Ž 0 1 . 0 0 0 6 6 - 6
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J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
synthesis, characteristics, and use of PAMAMdendrimers in respect to gene transfer into eukaryotic cells.
2. Transfection: overview and technologies Gene transfer into eukaryotic cells, or transfection, is a powerful tool for analysis of nucleic acid and protein function as well as for analysis of regulatory processes. This technique is especially important for analysis of the increasing number of genes of unknown function identified through genome sequencing projects. Gene transfer is also essential for biotechnology and gene therapy applications. The development of efficient transfection methods remains one of the major challenges in modern cell biology. 2.1. Gene transfer into eukaryotic cells Gene transfer into eukaryotic cells involves two key steps: Ž1. uptake of DNA by the cell; and Ž2. transport of DNA into the nucleus ŽFig. 1.. A transfection technology should therefore provide an efficient way for DNA to enter a cell, and then protect the DNA from the cell’s natural defense mechanisms until it has reached the nucleus. Depending on the application, the transfection technology should allow highly efficient gene transfer into either a broad range of cell types, or into a specific target cell type. Two main features of eukaryotic cells affect their ability to be transfected by exogenous DNA: the cell membrane and the intracellular transport and metabolic pathways. Different cell types have different lipid bilayer components, and hence different cell membrane characteristics. Features such as membrane fluidity, for example, appear to play a crucial role in DNA uptake. Similarly, different cell types possess different intracellular transport and metabolic pathways. Some types of epithelial cells, for example, have a very efficient system for uptake of extracellular molecules, and can therefore be easily transfected. In contrast, secreting cells Že.g. B cells, which produce antibodies. do not have a system for uptake of extra-
Fig. 1. Model of activated dendrimer-mediated DNA uptake. In the first step of the transfection process, the DNA᎐activated-dendrimer complex binds to the surface of the cell. The complex is then taken into the cell by endocytosis, and incorporated into the endosome of the cell. From the endosome the DNA is released into the cytosol. A small percentage of the released DNA reaches the nucleus, where it is transcribed into RNA. In the last step the RNA is transported back into the cytosol and then translated into protein. The exact pathway and metabolism of transfection reagents after release into the cytosol are still unclear.
cellular molecules, making gene transfer difficult. The ideal transfection technology would allow efficient transfection of all cell types, despite their differing intrinsic characteristics. There are also applications where specific transfection of only certain cell types is desirable. For example in gene therapy, specific transfection of the target tissue or cells avoids unwanted side effects in other tissues. The individual characteristics of different cell types play an important role in the establishment of transfection technologies for such applications. Thus, an ideal transfection technology would also provide flexibility in its cell specificity.
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
2.2. Classical transfection technologies Over the last 35 years a number of transfection technologies have been developed. Classical technologies include the use of infectious viral vectors, direct introduction of nucleic acids, and the use of chemicals to create DNA complexes that attach to the cell surface and are taken up by endocytosis. Viral vectors take advantage of the intrinsic ability of viruses to introduce nucleic acids into cells. The viral genome is manipulated such that non-essential endogenous sequences are replaced by an exogenous gene, and the resulting recombinant virus is used to infect the cells of interest. Viral vectors are highly specific for their host cells, and provide high transfection efficiencies. However, the generation of recombinant viruses is time-consuming and the amount of exogenous material that can be inserted into the vector is limited by the physical properties of the viral vector. In addition, viruses are not always useful for in vivo applications Že.g. gene therapy. due to their high immunogenicity and consequent induction of an immune response in host organisms. There is also the risk that endogenous viral-like sequences in the host cell genome will be activated by the recombinant virus, leading to the undesirable generation of new infectious viral particles with unknown properties. Nucleic acids can be directly introduced into cells by electroporation or microinjection. Microinjection, where DNA is injected into the cell nucleus using very fine needles, is useful for studying individual cultured cells. However, it is a highly skilled and laborious procedure where only a relatively low number of cells can be transfected, and furthermore, it is not suitable for all cell types. In electroporation, cells are subjected to a short high-voltage electrical pulse that forms pores in their cellular membrane through which macromolecules such as DNA can enter ŽWong and Neumann, 1982; Neumann et al., 1982.. This technique is more versatile than microinjection, but suffers from cytotoxicity and is not suitable for in vivo applications. The use of chemicals to generate DNA complexes that attach to the cell surface and are
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taken up by endocytosis was an early development in transfection technology. The first chemicals used were diethylaminoethyl ŽDEAE.-dextran and calcium phosphate ŽVaheri and Pagano, 1965; Graham and van der Eb, 1973.. DEAEdextran is a positively charged molecule that interacts with the negatively charged phosphate backbone of DNA. DNA᎐DEAE-dextran complexes appear to adhere to the cell surface and be taken into the cell by endocytosis. Similarly, DNA᎐calcium phosphate precipitates, generated by mixing DNA with calcium chloride in a phosphate buffer, also adhere to the cell surface and are taken into the cell by endocytosis. These methods have the major drawbacks of high cytotoxicity, lack of reproducibility, and limited applicability. 2.3. Modern transfection technologies Modern transfection technologies use optimized compounds to generate DNA complexes for transfection. Many modern transfection technologies are based on cationic liposomes ŽFelgner et al., 1987.. Transfection complexes form by interaction between positively charged liposomes with the negatively charged phosphate backbone of DNA. The transfection complexes can bind to the cell surface and be taken into the cell by endocytosis. Liposome technology often offers higher transfection efficiencies and better reproducibility than DEAE-dextran and calcium phosphate methods. However, liposome reagents are often cytotoxic, and transfection results can vary between different cell types ŽFigs. 2 and 3.. Activated PAMAM-dendrimers condense DNA into compact transfection complexes that can adhere to the cell surface and be taken into the cell via endocytosis ŽFig. 1., and offer significant advantages over other transfection technologies. Dendrimer-based reagents are less cytotoxic than many other transfection technologies ŽFig. 2., often provide higher transfection efficiencies than liposome-based reagents ŽFig. 3., and can be used with a broad range of cell types ŽHaensler and Szoka, 1993; see below.. Thus, activated PAMAM-dendrimers are very useful for cell biology. The remainder of this review will focus on acti-
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J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
PAMAM-dendrimers to be spherical. The terminal amine groups give PAMAM-dendrimers a net positive charge at physiological pH ŽpH 7᎐8.. At this pH, both protonized and unprotonized amine groups are present. Generation six or seven PAMAM-dendrimers with a diameter of 6᎐10 nm and a molecular mass of 30᎐50 kDa are generally used for gene transfer. 3.2. Acti¨ ation of PAMAM-dendrimers
Fig. 2. Low cytotoxicity of activated PAMAM-dendrimers. Transfection of HeLa-S3 cells using SuperFect Reagent ŽQIAGEN. and cationic Liposome L was performed according to the supplier’s recommendation. Post-transfection Ž48 h., the cells were analyzed by visual inspection for cytotoxic effects using phase-contrast microscopy. No detectable difference between untransfected cells Žc. and cells transfected with SuperFect Reagent Ža. could be observed. For cationic Liposome L Žb. cytotoxic effects Ži.e. dead cells. were observed.
Newly synthesized PAMAM-dendrimers have a defined size and shape. Solubilization in an appropriate solvolytic solvent and heating for a defined period of time leads to hydrolytic cleavage of some of the amido bonds in the inner part of the molecule and removal of some of the branches of the dendrimer ŽFig. 4.. Carboxyl groups form at the amido bond cleavage sites, and the molecular mass of the dendrimer is reduced by 20᎐25%. This process is called activation, and results in dendrimers with a higher degree of flexibility. Activation is a random process that gives a mixed population of dendrimer molecules differing slightly in molecular mass and structure. The
vated PAMAM-dendrimers and their use in gene transfer.
3. Activated PAMAM-dendrimers for gene transfer 3.1. Synthesis of PAMAM-dendrimers Dendrimers are spherical macromolecules that consist of a core moiety from which branches radiate ŽGreek: dendron, tree; Fig. 4.. The dendrimers relevant for gene transfer have a multifunctional amine as the core moiety, and are synthesized by repeated Michael Addition of methylacrylate and reaction of the product with ethylenediamine ŽHaensler and Szoka, 1993.. The resulting PAMAM-dendrimers have alternating amido and amine bonds and are built up by layers of ‘shells’. The different shells are called generations. After generation four, steric factors cause
Fig. 3. Comparison of transfection efficiencies obtained using SuperFect Reagent and four of the most commonly used liposome reagents. COS-7 and HeLa-S3 were transfected in 96-well plates with 0.5 g pCMV using SuperFect Reagent and four of the most commonly used liposome reagents. Cells Ž2 = 10 4 per well. were seeded 1 day prior to transfection. Transfections were performed according to the supplier’s recommendation. Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
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Fig. 4. Non-activated and activated PAMAM-dendrimers. Schematic diagram of a non-activated Žleft. and activated dendrimer Žmiddle.. The right panel shows a magnification of the dendrimer branches.
overall size and shape of the dendrimer molecule, however, does not change following activation. Both non-activated and activated dendrimers interact electrostatically with DNA to form DNA᎐dendrimer complexes that mediate gene transfer Žsee below.. However, in cultured eukaryotic cells, the transfection efficiency of activated dendrimers is 2᎐3 orders of magnitudes higher than that of non-activated dendrimers ŽTang et al., 1996.. This appears to be due to the higher flexibility of activated dendrimers compared to the more rigid structure of newly synthesized non-activated dendrimers. This increased flexibility may play a key role in the release of DNA from the endosome, as discussed below. 3.3. Gene transfer mediated by acti¨ ated PAMAMdendrimers For transfection, activated PAMAM-dendrimers are mixed with the DNA of interest. Positively charged amino groups on the surface of the dendrimer molecule interact with the negatively charged phosphate groups of the DNA molecule to form a DNA᎐dendrimer complex with a toroid-like structure ŽFig. 5.. DNA molecules are highly condensed within the complex ŽTang et al., 1996.: a 6-kb plasmid, for example, has an extended structure several hundred nanometers in diameter, while a DNA᎐den-
drimer complex, which contains several DNA molecules, has a mean diameter of 50᎐100 nm ŽTang and Szoka, 1997.. For gene transfer experiments in cell culture, an 8᎐12-fold excess of positive amino groups over negative phosphate groups is usually used. Under these conditions the DNA᎐dendrimer complex has a positive net charge and can bind to negatively charged surface molecules on the membrane of eukaryotic cells. Complexes bound to the cell surface are taken into the cell by non-specific endocytosis. Once inside the cell, the complexes are transported to the endosomes. The properties and behavior of DNA᎐dendrimer complexes within the endosomes appear to be a crucial part of the transfection process ŽTang et al., 1996.. DNA is protected from degradation by endosomal nucleases by being highly condensed within the DNA᎐dendrimer complex. In addition, amino groups on the dendrimers that are unprotonated at neutral pH can become protonated in the acidic environment of the endosome. This leads to buffering of the endosome, which inhibits pH-dependent endosomal nucleases. In the model proposed by Tang et al. Ž1996., the buffering properties of the dendrimer also appear to be important for release of DNA from the endosome. Activated dendrimers are proposed to have a fully extended conformation at
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J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
DNA or transfection complex is transported into the nucleus remains to be elucidated. 3.4. Parameters influencing transfection with PAMAM-dendrimers
Fig. 5. Model of the toroid-like complex between plasmid DNA and dendrimers. Dendrimer molecules Žspheres. are located both inside and outside the coiled DNA molecules, and more than one plasmid DNA molecule can be present in one toroid-like complex. The left part of the graphic shows the most probable situation; the dendrimers on the outside of the complex are not shown in the remainder of the graphic in order to give a better view of the inner part of the complex. The diameter of such a complex is approximately 50᎐100 nm.
neutral pH due to electrostatic repulsion between protonated primary amines at the branch termini. Charge neutralization of these terminal groups by DNA would cause the dendrimers to collapse into a compact form in the dendrimer᎐DNA complex. In the endosome, protonation of interior tertiary amines would increase the positive charge of the dendrimer, meaning that fewer dendrimer molecules would be required to maintain charge neutralization of the DNA and that excess dendrimer molecules would be released from the complex. The released dendrimers would increase in volume as they returned to a fully hydrated form, leading to swelling and eventual lysis of the endosome, and release of the DNA Žeither free or still as a complex with the dendrimers. into the cytosol. This model may explain the observation that activated dendrimers provide higher transfection efficiencies than non-activated dendrimers: the more rigid structure of non-activated dendrimers may prevent them from contracting in the DNA᎐dendrimer complex and swelling in the endosome, and hence DNA or transfection complexes may not be released as efficiently into the cytosol. The mechanism by which the released
Two main factors influence the transfection efficiency and cell specificity of different PAMAM-dendrimers: the generation number and the activation time. The generation number determines the size of the dendrimer molecule, which may influence the nature of the DNA᎐dendrimer complex ŽHaensler and Szoka, 1993.. As described above, activation involves the random removal of branches from the newly synthesized dendrimer molecule and results in a more flexible molecule that provides more efficient transfection. If the reaction time is too short, then the dendrimer will not be flexible enough for efficient transfection. Conversely if the activation reaction proceeds for too long, the dendrimers will lack sufficient charge density to form a complex with DNA andror lack sufficient mass to swell and rupture the endosome ŽTang et al., 1996.. As yet it is not possible to predict theoretically the transfection characteristics of an activated PAMAMdendrimer. Thus, both optimal generation number and optimal activation time must be determined empirically. Optimization experiments are typically performed using reporter gene plasmids, which encode an enzyme whose activity can be easily assayed, to monitor transfection efficiency. Successful transfection results in synthesis of the enzyme in transfected cells, with the amount of enzyme synthesized Žand hence enzyme activity. correlating with the transfection efficiency. An example of an optimization experiment for dendrimer generation number using NIHr3T3 cells Ža mouse fibroblastic cell line. is shown in Fig. 6. In this experiment, cells were transfected with a galactosidase expression plasmid using activated PAMAM-dendrimers of generation five, six and seven. The highest level of -galactosidase activity, and hence the highest transfection efficiency, was observed using generation six dendrimers. Generation seven dendrimers showed a twofold lower efficiency, while generation five dendrimers
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
Fig. 6. Influence of dendrimer generation on transfection efficiency. NIHr3T3 cells, a mouse fibroblastic cell line, were plated 24 h before transfection in 96-well cell culture plates at a density of 2 = 10 4 cellsrwell. For transfection, DNA and the different dendrimer solutions Žactivated dendrimers of generation 5, 6, or 7. were diluted separately in cell culture medium ŽDMEM. without serum and antibiotics Ž0.5 g pCMV plasmid DNA in a volume of 30 lrwell and 9 g or 12 g activated dendrimer in a volume of 20 l.. Diluted DNA was mixed with each diluted dendrimer solution, and incubated for 10 min at room temperature. The cells were washed once with PBS. Cell culture medium Ž100 l. with serum and antibiotics was added to each DNA᎐dendrimer mix, and the complex solutions were transferred onto the cells. After addition of the transfection complexes Ž3 h., the medium was removed, the cells were washed once with PBS, and then fresh medium containing serum and antibiotics was added. After transfection Ž48 h., the cells were lysed and assayed for -galactosidase activity. The highest -galactosidase activity Žcorresponding to the highest transfection efficiency. was observed using generation six dendrimers. Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
showed an eight to ninefold lower efficiency. This demonstrates that NIHr3T3 cells require dendrimers of a certain size for optimal gene transfer. An example of an optimization experiment for dendrimer activation time is shown in Fig. 7. NIHr3T3 cells were transfected with the same -galactosidase expression plasmid using PAMAM-dendrimers that differed in their activation time, from no activation Ž t 0 . to increasing activation times Ž t 1 ᎐t5 .. Non-activated dendrimers achieved only a low transfection efficiency. Transfection efficiency increased as activation time increased from t 1 to t 3 , with the highest transfection efficiency observed using dendrimers of activation time t 3 . Transfection efficiency decreased
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with longer activation times, and was almost completely abrogated using dendrimers with an activation time of t5 . These results demonstrate the importance of activation for efficient gene transfer. The generation number and activation time that give the highest transfection efficiency can vary between different cell types andror different applications Že.g. the type of nucleic acid transfected or in vivo vs. in vitro applications.. This means that for optimal results using different cell types and different applications, it may be necessary to optimize generation number and activation time. Other parameters that need to be optimized for efficient transfection and minimal cytotoxicity are the amounts of activated dendrimer and nucleic acid used for transfection and the ratio of dendrimer to DNA. In addition, PAMAM-dendrimers can be synthesized using different core moieties. The ability to produce a range of different PAMAM-dendrimers that differ in their chemical properties Ži.e. core moiety, generation number, and activation time. provides tremendous scope for developing dendrimer-based transfection reagents for a broad range of cell types and for different applications. This versatility, together with high transfection efficiencies, low
Fig. 7. Influence of dendrimer activation on transfection efficiency. NIHr3T3 cells were transfected as described in the legend of Fig. 6. Instead of dendrimers of different generation, dendrimers were used that differed in their activation time. t 0 : non-activated PAMAM-dendrimer. pCMV-DNA Ž0.5 g. and 3, 6 and 9 g of the dendrimer were used in transfection experiments. The highest transfection efficiency was observed using dendrimers of activation time t 3 . Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
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cytotoxicity, and defined size and shape through a controlled synthesis process, makes activated PAMAM-dendrimers a powerful transfection technology. 3.5. Efficient transfection of different cell types with PAMAM-dendrimers The biotechnology company QIAGEN has developed two commercially available transfection reagents based on activated PAMAM-dendrimers ᎏ SuperFect 䊛 Transfection Reagent and PolyFect 䊛 Transfection Reagent. These two reagents differ in their core moiety, generation number, and activation procedure. SuperFect Reagent, introduced in 1997, and PolyFect Reagent, introduced in 2000, have been successfully used in many laboratories for gene transfer experiments in different cell types. These include fibroblast and epithelial cells Že.g. Albritton, 1997., neuronal cell lines ŽDaniels et al., 1998., T cell lines ŽDenisenko and Bomsztyk, 1997., primary aortic smooth muscle cells ŽRafty and Khachigian, 1998. and mouse embryo fibroblasts ŽKamijo et al., 1997.. Activated dendrimers have also been used successfully for transfection of recombinant adenoviral vectors ŽTang et al., 1997. and baculovirus in insect cells ŽZandi et al., 1998..
4. Conclusions and prospects Despite the success of activated PAMAM-dendrimers in gene transfer experiments there are many challenges left for the future. Some cell types are very difficult to transfect. These include primary cells Žcells obtained directly from tissue, that are only capable of a limited number of generations before the population senesces and that possess characteristics similar to in vivo cells., continuous cell lines derived from B cells and T cells, non-dividing cells, and many others. Because of their versatility, activated PAMAM-dendrimers could provide solutions for these cell types. In the future, in vivo gene transfer Ži.e. gene transfer into multicellular organisms rather than
cultured cells. will become more and more important for biology and medicine. Gene therapy strategies, in which functional genes are used to treat genetic diseases, require a non-toxic and highly efficient transfection technology. Several problems remain to be solved for such applications. For example, the stability of the transfection complex, the influence of serum proteins and the extracellular matrix on the transfection complex, and problems with the diffusion of the transfection complex through tissues are important considerations that have not yet been fully optimized for many transfection technologies. In addition, specific transfection of only certain cell populations is often desirable in gene therapy strategies. At present, this is mainly addressed by designing therapeutic DNA that can only be expressed in the target cells. However, it is possible that a highly cell-specific transfection technology could also be developed. Much work is needed to develop transfection reagents based on activated PAMAM-dendrimers that fulfill all criteria for efficient in vivo transfection. Cell specificity is one of the most important problems facing the use of PAMAM-dendrimers in this application, and it may even be necessary to combine activated PAMAM-dendrimers with other technologies to provide this. Nevertheless, the advantage of current activated PAMAM-dendrimers over other transfection technologies, together with their versatility, suggests that dendrimers will be one of the technologies that provides solutions to these remaining transfection problems.
Acknowledgements The authors wish to thank Drs Ute Kruger, ¨ Martin Weber, and Christoph Erbacher for helpful comments regarding the manuscript. References Albritton, L.M., 1997. Efficient transfection of fibroblast and epithelial cells using an activated dendrimer reagent. J. NIH Res. 9, 52.
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347 Daniels, R.H., Hall, P.S., Bokoch, G.M., 1998. Membrane targeting of p21-activated kinase 1 ŽPAK1. induces neurite outgrowth from PC-12 cells. EMBO J. 17, 754᎐764. Denisenko, O.N., Bomsztyk, K., 1997. The product of the murine homolog of the Drosophila extra sex combs gene displays transcriptional repressor activity. Mol. Cell. Biol. 17, 4707᎐4717. Felgner, P.L., Gadek, T.R., Holm, M. et al., 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA 84, 7413᎐7417. Graham, F.L., van der Eb, A.J., 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456᎐467. Haensler, J., Szoka, F.C., 1993. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 4, 372᎐379. Kamijo, T., Zindy, F., Roussel, M.F. et al., 1997. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649᎐659. Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P.H., 1982. Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 1, 841᎐845.
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Rafty, L.A., Khachigian, L.M., 1998. Zinc finger transcription factors mediate high constitutive platelet-derived growth factor-B expression in smooth muscle cells derived from aortae of newborn rats. J. Biol. Chem. 273, 5758᎐5764. Tang, M.X., Szoka Jr., F.C., 1997. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Therapy 4, 823᎐832. Tang, M.X., Redemann, C.T., Szoka Jr., F.C., 1996. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjugate Chem. 7, 703᎐714. Tang, D.C., Jennelle, R.S., Shi, Z. et al., 1997. Overexpression of adenovirus-encoded transgenes from the cytomegalovirus immediate early promoter in irradiated tumor cells. Hum. Gene Ther. 8, 2117᎐2124. Vaheri, A., Pagano, J.S., 1965. Infectious poliovirus RNA: a sensitive method of assay. Virology 27, 434᎐436. Wong, T.K., Neumann, E., 1982. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584᎐587. Zandi, E., Chen, Y., Karin, M., 1998. Direct phosphorylation of IkappaB by IKKalpha and IKKbeta: discrimination between free and NF-kappaB-bound substrate. Science 281, 1360᎐1363.
Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers Jorg ¨ DennigU , Emma Duncan QIAGEN GmbH, Max-Volmer Straße 4, 40724 Hilden, Germany
Abstract The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency andror restricted applicability. Activated polyamidoamine ŽPAMAM.-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications. 䊚 2002 Elsevier Science B.V. All rights reserved. Keywords: Polyamidoamine dendrimers; Transfection
1. Introduction Gene transfer into eukaryotic cells Žtransfection. is a useful tool for molecular biotechnology. Over the last 35 years a number of different transfection technologies have been developed. Classical technologies include the use of infectious viral vectors, direct introduction of nucleic acids by electroporation or microinjection, and the use of chemicals to create DNA complexes
U
Corresponding author. Fax: q49-2103-29-26-326. E-mail address: [email protected] ŽJ. Dennig..
that attach to the cell surface and are taken up by endocytosis. Each of these approaches has associated disadvantages, the most problematic being cytotoxicity and low transfection efficiency. Activated polyamidoamine ŽPAMAM.-dendrimers provide a new transfection technology that offers significant advantages over classical transfection methods as well as cationic liposomes. Transfection reagents based on this technology provide high transfection efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. In this review we first give a brief overview of transfection and existing transfection technologies, and then discuss the
1389-0352r02r$ - see front matter 䊚 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 9 - 0 3 5 2 Ž 0 1 . 0 0 0 6 6 - 6
340
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
synthesis, characteristics, and use of PAMAMdendrimers in respect to gene transfer into eukaryotic cells.
2. Transfection: overview and technologies Gene transfer into eukaryotic cells, or transfection, is a powerful tool for analysis of nucleic acid and protein function as well as for analysis of regulatory processes. This technique is especially important for analysis of the increasing number of genes of unknown function identified through genome sequencing projects. Gene transfer is also essential for biotechnology and gene therapy applications. The development of efficient transfection methods remains one of the major challenges in modern cell biology. 2.1. Gene transfer into eukaryotic cells Gene transfer into eukaryotic cells involves two key steps: Ž1. uptake of DNA by the cell; and Ž2. transport of DNA into the nucleus ŽFig. 1.. A transfection technology should therefore provide an efficient way for DNA to enter a cell, and then protect the DNA from the cell’s natural defense mechanisms until it has reached the nucleus. Depending on the application, the transfection technology should allow highly efficient gene transfer into either a broad range of cell types, or into a specific target cell type. Two main features of eukaryotic cells affect their ability to be transfected by exogenous DNA: the cell membrane and the intracellular transport and metabolic pathways. Different cell types have different lipid bilayer components, and hence different cell membrane characteristics. Features such as membrane fluidity, for example, appear to play a crucial role in DNA uptake. Similarly, different cell types possess different intracellular transport and metabolic pathways. Some types of epithelial cells, for example, have a very efficient system for uptake of extracellular molecules, and can therefore be easily transfected. In contrast, secreting cells Že.g. B cells, which produce antibodies. do not have a system for uptake of extra-
Fig. 1. Model of activated dendrimer-mediated DNA uptake. In the first step of the transfection process, the DNA᎐activated-dendrimer complex binds to the surface of the cell. The complex is then taken into the cell by endocytosis, and incorporated into the endosome of the cell. From the endosome the DNA is released into the cytosol. A small percentage of the released DNA reaches the nucleus, where it is transcribed into RNA. In the last step the RNA is transported back into the cytosol and then translated into protein. The exact pathway and metabolism of transfection reagents after release into the cytosol are still unclear.
cellular molecules, making gene transfer difficult. The ideal transfection technology would allow efficient transfection of all cell types, despite their differing intrinsic characteristics. There are also applications where specific transfection of only certain cell types is desirable. For example in gene therapy, specific transfection of the target tissue or cells avoids unwanted side effects in other tissues. The individual characteristics of different cell types play an important role in the establishment of transfection technologies for such applications. Thus, an ideal transfection technology would also provide flexibility in its cell specificity.
J. Dennig, E. Duncan r Re¨ iews in Molecular Biotechnology 90 (2002) 339᎐347
2.2. Classical transfection technologies Over the last 35 years a number of transfection technologies have been developed. Classical technologies include the use of infectious viral vectors, direct introduction of nucleic acids, and the use of chemicals to create DNA complexes that attach to the cell surface and are taken up by endocytosis. Viral vectors take advantage of the intrinsic ability of viruses to introduce nucleic acids into cells. The viral genome is manipulated such that non-essential endogenous sequences are replaced by an exogenous gene, and the resulting recombinant virus is used to infect the cells of interest. Viral vectors are highly specific for their host cells, and provide high transfection efficiencies. However, the generation of recombinant viruses is time-consuming and the amount of exogenous material that can be inserted into the vector is limited by the physical properties of the viral vector. In addition, viruses are not always useful for in vivo applications Že.g. gene therapy. due to their high immunogenicity and consequent induction of an immune response in host organisms. There is also the risk that endogenous viral-like sequences in the host cell genome will be activated by the recombinant virus, leading to the undesirable generation of new infectious viral particles with unknown properties. Nucleic acids can be directly introduced into cells by electroporation or microinjection. Microinjection, where DNA is injected into the cell nucleus using very fine needles, is useful for studying individual cultured cells. However, it is a highly skilled and laborious procedure where only a relatively low number of cells can be transfected, and furthermore, it is not suitable for all cell types. In electroporation, cells are subjected to a short high-voltage electrical pulse that forms pores in their cellular membrane through which macromolecules such as DNA can enter ŽWong and Neumann, 1982; Neumann et al., 1982.. This technique is more versatile than microinjection, but suffers from cytotoxicity and is not suitable for in vivo applications. The use of chemicals to generate DNA complexes that attach to the cell surface and are
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taken up by endocytosis was an early development in transfection technology. The first chemicals used were diethylaminoethyl ŽDEAE.-dextran and calcium phosphate ŽVaheri and Pagano, 1965; Graham and van der Eb, 1973.. DEAEdextran is a positively charged molecule that interacts with the negatively charged phosphate backbone of DNA. DNA᎐DEAE-dextran complexes appear to adhere to the cell surface and be taken into the cell by endocytosis. Similarly, DNA᎐calcium phosphate precipitates, generated by mixing DNA with calcium chloride in a phosphate buffer, also adhere to the cell surface and are taken into the cell by endocytosis. These methods have the major drawbacks of high cytotoxicity, lack of reproducibility, and limited applicability. 2.3. Modern transfection technologies Modern transfection technologies use optimized compounds to generate DNA complexes for transfection. Many modern transfection technologies are based on cationic liposomes ŽFelgner et al., 1987.. Transfection complexes form by interaction between positively charged liposomes with the negatively charged phosphate backbone of DNA. The transfection complexes can bind to the cell surface and be taken into the cell by endocytosis. Liposome technology often offers higher transfection efficiencies and better reproducibility than DEAE-dextran and calcium phosphate methods. However, liposome reagents are often cytotoxic, and transfection results can vary between different cell types ŽFigs. 2 and 3.. Activated PAMAM-dendrimers condense DNA into compact transfection complexes that can adhere to the cell surface and be taken into the cell via endocytosis ŽFig. 1., and offer significant advantages over other transfection technologies. Dendrimer-based reagents are less cytotoxic than many other transfection technologies ŽFig. 2., often provide higher transfection efficiencies than liposome-based reagents ŽFig. 3., and can be used with a broad range of cell types ŽHaensler and Szoka, 1993; see below.. Thus, activated PAMAM-dendrimers are very useful for cell biology. The remainder of this review will focus on acti-
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PAMAM-dendrimers to be spherical. The terminal amine groups give PAMAM-dendrimers a net positive charge at physiological pH ŽpH 7᎐8.. At this pH, both protonized and unprotonized amine groups are present. Generation six or seven PAMAM-dendrimers with a diameter of 6᎐10 nm and a molecular mass of 30᎐50 kDa are generally used for gene transfer. 3.2. Acti¨ ation of PAMAM-dendrimers
Fig. 2. Low cytotoxicity of activated PAMAM-dendrimers. Transfection of HeLa-S3 cells using SuperFect Reagent ŽQIAGEN. and cationic Liposome L was performed according to the supplier’s recommendation. Post-transfection Ž48 h., the cells were analyzed by visual inspection for cytotoxic effects using phase-contrast microscopy. No detectable difference between untransfected cells Žc. and cells transfected with SuperFect Reagent Ža. could be observed. For cationic Liposome L Žb. cytotoxic effects Ži.e. dead cells. were observed.
Newly synthesized PAMAM-dendrimers have a defined size and shape. Solubilization in an appropriate solvolytic solvent and heating for a defined period of time leads to hydrolytic cleavage of some of the amido bonds in the inner part of the molecule and removal of some of the branches of the dendrimer ŽFig. 4.. Carboxyl groups form at the amido bond cleavage sites, and the molecular mass of the dendrimer is reduced by 20᎐25%. This process is called activation, and results in dendrimers with a higher degree of flexibility. Activation is a random process that gives a mixed population of dendrimer molecules differing slightly in molecular mass and structure. The
vated PAMAM-dendrimers and their use in gene transfer.
3. Activated PAMAM-dendrimers for gene transfer 3.1. Synthesis of PAMAM-dendrimers Dendrimers are spherical macromolecules that consist of a core moiety from which branches radiate ŽGreek: dendron, tree; Fig. 4.. The dendrimers relevant for gene transfer have a multifunctional amine as the core moiety, and are synthesized by repeated Michael Addition of methylacrylate and reaction of the product with ethylenediamine ŽHaensler and Szoka, 1993.. The resulting PAMAM-dendrimers have alternating amido and amine bonds and are built up by layers of ‘shells’. The different shells are called generations. After generation four, steric factors cause
Fig. 3. Comparison of transfection efficiencies obtained using SuperFect Reagent and four of the most commonly used liposome reagents. COS-7 and HeLa-S3 were transfected in 96-well plates with 0.5 g pCMV using SuperFect Reagent and four of the most commonly used liposome reagents. Cells Ž2 = 10 4 per well. were seeded 1 day prior to transfection. Transfections were performed according to the supplier’s recommendation. Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
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Fig. 4. Non-activated and activated PAMAM-dendrimers. Schematic diagram of a non-activated Žleft. and activated dendrimer Žmiddle.. The right panel shows a magnification of the dendrimer branches.
overall size and shape of the dendrimer molecule, however, does not change following activation. Both non-activated and activated dendrimers interact electrostatically with DNA to form DNA᎐dendrimer complexes that mediate gene transfer Žsee below.. However, in cultured eukaryotic cells, the transfection efficiency of activated dendrimers is 2᎐3 orders of magnitudes higher than that of non-activated dendrimers ŽTang et al., 1996.. This appears to be due to the higher flexibility of activated dendrimers compared to the more rigid structure of newly synthesized non-activated dendrimers. This increased flexibility may play a key role in the release of DNA from the endosome, as discussed below. 3.3. Gene transfer mediated by acti¨ ated PAMAMdendrimers For transfection, activated PAMAM-dendrimers are mixed with the DNA of interest. Positively charged amino groups on the surface of the dendrimer molecule interact with the negatively charged phosphate groups of the DNA molecule to form a DNA᎐dendrimer complex with a toroid-like structure ŽFig. 5.. DNA molecules are highly condensed within the complex ŽTang et al., 1996.: a 6-kb plasmid, for example, has an extended structure several hundred nanometers in diameter, while a DNA᎐den-
drimer complex, which contains several DNA molecules, has a mean diameter of 50᎐100 nm ŽTang and Szoka, 1997.. For gene transfer experiments in cell culture, an 8᎐12-fold excess of positive amino groups over negative phosphate groups is usually used. Under these conditions the DNA᎐dendrimer complex has a positive net charge and can bind to negatively charged surface molecules on the membrane of eukaryotic cells. Complexes bound to the cell surface are taken into the cell by non-specific endocytosis. Once inside the cell, the complexes are transported to the endosomes. The properties and behavior of DNA᎐dendrimer complexes within the endosomes appear to be a crucial part of the transfection process ŽTang et al., 1996.. DNA is protected from degradation by endosomal nucleases by being highly condensed within the DNA᎐dendrimer complex. In addition, amino groups on the dendrimers that are unprotonated at neutral pH can become protonated in the acidic environment of the endosome. This leads to buffering of the endosome, which inhibits pH-dependent endosomal nucleases. In the model proposed by Tang et al. Ž1996., the buffering properties of the dendrimer also appear to be important for release of DNA from the endosome. Activated dendrimers are proposed to have a fully extended conformation at
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DNA or transfection complex is transported into the nucleus remains to be elucidated. 3.4. Parameters influencing transfection with PAMAM-dendrimers
Fig. 5. Model of the toroid-like complex between plasmid DNA and dendrimers. Dendrimer molecules Žspheres. are located both inside and outside the coiled DNA molecules, and more than one plasmid DNA molecule can be present in one toroid-like complex. The left part of the graphic shows the most probable situation; the dendrimers on the outside of the complex are not shown in the remainder of the graphic in order to give a better view of the inner part of the complex. The diameter of such a complex is approximately 50᎐100 nm.
neutral pH due to electrostatic repulsion between protonated primary amines at the branch termini. Charge neutralization of these terminal groups by DNA would cause the dendrimers to collapse into a compact form in the dendrimer᎐DNA complex. In the endosome, protonation of interior tertiary amines would increase the positive charge of the dendrimer, meaning that fewer dendrimer molecules would be required to maintain charge neutralization of the DNA and that excess dendrimer molecules would be released from the complex. The released dendrimers would increase in volume as they returned to a fully hydrated form, leading to swelling and eventual lysis of the endosome, and release of the DNA Žeither free or still as a complex with the dendrimers. into the cytosol. This model may explain the observation that activated dendrimers provide higher transfection efficiencies than non-activated dendrimers: the more rigid structure of non-activated dendrimers may prevent them from contracting in the DNA᎐dendrimer complex and swelling in the endosome, and hence DNA or transfection complexes may not be released as efficiently into the cytosol. The mechanism by which the released
Two main factors influence the transfection efficiency and cell specificity of different PAMAM-dendrimers: the generation number and the activation time. The generation number determines the size of the dendrimer molecule, which may influence the nature of the DNA᎐dendrimer complex ŽHaensler and Szoka, 1993.. As described above, activation involves the random removal of branches from the newly synthesized dendrimer molecule and results in a more flexible molecule that provides more efficient transfection. If the reaction time is too short, then the dendrimer will not be flexible enough for efficient transfection. Conversely if the activation reaction proceeds for too long, the dendrimers will lack sufficient charge density to form a complex with DNA andror lack sufficient mass to swell and rupture the endosome ŽTang et al., 1996.. As yet it is not possible to predict theoretically the transfection characteristics of an activated PAMAMdendrimer. Thus, both optimal generation number and optimal activation time must be determined empirically. Optimization experiments are typically performed using reporter gene plasmids, which encode an enzyme whose activity can be easily assayed, to monitor transfection efficiency. Successful transfection results in synthesis of the enzyme in transfected cells, with the amount of enzyme synthesized Žand hence enzyme activity. correlating with the transfection efficiency. An example of an optimization experiment for dendrimer generation number using NIHr3T3 cells Ža mouse fibroblastic cell line. is shown in Fig. 6. In this experiment, cells were transfected with a galactosidase expression plasmid using activated PAMAM-dendrimers of generation five, six and seven. The highest level of -galactosidase activity, and hence the highest transfection efficiency, was observed using generation six dendrimers. Generation seven dendrimers showed a twofold lower efficiency, while generation five dendrimers
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Fig. 6. Influence of dendrimer generation on transfection efficiency. NIHr3T3 cells, a mouse fibroblastic cell line, were plated 24 h before transfection in 96-well cell culture plates at a density of 2 = 10 4 cellsrwell. For transfection, DNA and the different dendrimer solutions Žactivated dendrimers of generation 5, 6, or 7. were diluted separately in cell culture medium ŽDMEM. without serum and antibiotics Ž0.5 g pCMV plasmid DNA in a volume of 30 lrwell and 9 g or 12 g activated dendrimer in a volume of 20 l.. Diluted DNA was mixed with each diluted dendrimer solution, and incubated for 10 min at room temperature. The cells were washed once with PBS. Cell culture medium Ž100 l. with serum and antibiotics was added to each DNA᎐dendrimer mix, and the complex solutions were transferred onto the cells. After addition of the transfection complexes Ž3 h., the medium was removed, the cells were washed once with PBS, and then fresh medium containing serum and antibiotics was added. After transfection Ž48 h., the cells were lysed and assayed for -galactosidase activity. The highest -galactosidase activity Žcorresponding to the highest transfection efficiency. was observed using generation six dendrimers. Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
showed an eight to ninefold lower efficiency. This demonstrates that NIHr3T3 cells require dendrimers of a certain size for optimal gene transfer. An example of an optimization experiment for dendrimer activation time is shown in Fig. 7. NIHr3T3 cells were transfected with the same -galactosidase expression plasmid using PAMAM-dendrimers that differed in their activation time, from no activation Ž t 0 . to increasing activation times Ž t 1 ᎐t5 .. Non-activated dendrimers achieved only a low transfection efficiency. Transfection efficiency increased as activation time increased from t 1 to t 3 , with the highest transfection efficiency observed using dendrimers of activation time t 3 . Transfection efficiency decreased
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with longer activation times, and was almost completely abrogated using dendrimers with an activation time of t5 . These results demonstrate the importance of activation for efficient gene transfer. The generation number and activation time that give the highest transfection efficiency can vary between different cell types andror different applications Že.g. the type of nucleic acid transfected or in vivo vs. in vitro applications.. This means that for optimal results using different cell types and different applications, it may be necessary to optimize generation number and activation time. Other parameters that need to be optimized for efficient transfection and minimal cytotoxicity are the amounts of activated dendrimer and nucleic acid used for transfection and the ratio of dendrimer to DNA. In addition, PAMAM-dendrimers can be synthesized using different core moieties. The ability to produce a range of different PAMAM-dendrimers that differ in their chemical properties Ži.e. core moiety, generation number, and activation time. provides tremendous scope for developing dendrimer-based transfection reagents for a broad range of cell types and for different applications. This versatility, together with high transfection efficiencies, low
Fig. 7. Influence of dendrimer activation on transfection efficiency. NIHr3T3 cells were transfected as described in the legend of Fig. 6. Instead of dendrimers of different generation, dendrimers were used that differed in their activation time. t 0 : non-activated PAMAM-dendrimer. pCMV-DNA Ž0.5 g. and 3, 6 and 9 g of the dendrimer were used in transfection experiments. The highest transfection efficiency was observed using dendrimers of activation time t 3 . Transfection efficiencies are given as -galactosidase unitsrml. Each bar represents the average efficiency from four replicates.
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cytotoxicity, and defined size and shape through a controlled synthesis process, makes activated PAMAM-dendrimers a powerful transfection technology. 3.5. Efficient transfection of different cell types with PAMAM-dendrimers The biotechnology company QIAGEN has developed two commercially available transfection reagents based on activated PAMAM-dendrimers ᎏ SuperFect 䊛 Transfection Reagent and PolyFect 䊛 Transfection Reagent. These two reagents differ in their core moiety, generation number, and activation procedure. SuperFect Reagent, introduced in 1997, and PolyFect Reagent, introduced in 2000, have been successfully used in many laboratories for gene transfer experiments in different cell types. These include fibroblast and epithelial cells Že.g. Albritton, 1997., neuronal cell lines ŽDaniels et al., 1998., T cell lines ŽDenisenko and Bomsztyk, 1997., primary aortic smooth muscle cells ŽRafty and Khachigian, 1998. and mouse embryo fibroblasts ŽKamijo et al., 1997.. Activated dendrimers have also been used successfully for transfection of recombinant adenoviral vectors ŽTang et al., 1997. and baculovirus in insect cells ŽZandi et al., 1998..
4. Conclusions and prospects Despite the success of activated PAMAM-dendrimers in gene transfer experiments there are many challenges left for the future. Some cell types are very difficult to transfect. These include primary cells Žcells obtained directly from tissue, that are only capable of a limited number of generations before the population senesces and that possess characteristics similar to in vivo cells., continuous cell lines derived from B cells and T cells, non-dividing cells, and many others. Because of their versatility, activated PAMAM-dendrimers could provide solutions for these cell types. In the future, in vivo gene transfer Ži.e. gene transfer into multicellular organisms rather than
cultured cells. will become more and more important for biology and medicine. Gene therapy strategies, in which functional genes are used to treat genetic diseases, require a non-toxic and highly efficient transfection technology. Several problems remain to be solved for such applications. For example, the stability of the transfection complex, the influence of serum proteins and the extracellular matrix on the transfection complex, and problems with the diffusion of the transfection complex through tissues are important considerations that have not yet been fully optimized for many transfection technologies. In addition, specific transfection of only certain cell populations is often desirable in gene therapy strategies. At present, this is mainly addressed by designing therapeutic DNA that can only be expressed in the target cells. However, it is possible that a highly cell-specific transfection technology could also be developed. Much work is needed to develop transfection reagents based on activated PAMAM-dendrimers that fulfill all criteria for efficient in vivo transfection. Cell specificity is one of the most important problems facing the use of PAMAM-dendrimers in this application, and it may even be necessary to combine activated PAMAM-dendrimers with other technologies to provide this. Nevertheless, the advantage of current activated PAMAM-dendrimers over other transfection technologies, together with their versatility, suggests that dendrimers will be one of the technologies that provides solutions to these remaining transfection problems.
Acknowledgements The authors wish to thank Drs Ute Kruger, ¨ Martin Weber, and Christoph Erbacher for helpful comments regarding the manuscript. References Albritton, L.M., 1997. Efficient transfection of fibroblast and epithelial cells using an activated dendrimer reagent. J. NIH Res. 9, 52.
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