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Bioconjugate Chem. 2003, 14, 222−231
Novel Shielded Transferrin-Polyethylene Glycol-Polyethylenimine/ DNA Complexes for Systemic Tumor-Targeted Gene Transfer Malgorzata Kursa,† Greg F. Walker,‡ Vanessa Roessler,† Manfred Ogris,‡ Wolfgang Roedl,‡ Ralf Kircheis,†,§ and Ernst Wagner*,‡ Pharmaceutical Biology-Biotechnology, Department for Pharmacy, Ludwig-Maximilians-Universitaet, Butenandtstrasse 5-13, D-81377 Muenchen, Germany, and Boehringer Ingelheim Austria, Dr Boehringer Gasse 5-11, A-1121 Vienna, Austria. Received September 20, 2002; Revised Manuscript Received November 7, 2002
Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-R), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freezethawing.
INTRODUCTION
The ability of nonviral DNA complexes to deliver genes specifically to cells and tissues in vivo offers a potential therapy for many diseases that are currently considered incurable, including some disseminated tumors (1). To date, the main developmental focus for nonviral DNA complexes for systemic tumor targeting has been improving their gene transfer in vivo. Beside the need to improve delivery efficiency, the formulation of well-defined, easily prepared complexes that can be stored over long periods is also necessary for their practical administration and development as a pharmaceutical product. For tumorspecific uptake, polyplexes (2) are usually made in relatively small batches and are required to be freshly prepared before use as they form large aggregates when stored by freezing or freeze-drying (3, 4). Furthermore, nonviral-based gene delivery systems have to be administered many times, and the formation of many types of DNA complexes requires sequential reaction steps which are both laborious and inconvenient. Polyplexes are formed by the mixing of a solution of plasmid DNA with a solution of a cationic polymer which * Corresponding author. Telephone: ++49-89-218077841. Fax: ++49-89-2180-77798. E-mail: ernst.wagner@ cup.uni-muenchen.de. † Boehringer Ingelheim Austria. ‡ Ludwig-Maximilians-University, Muenchen. § Present address: Igeneon AG, Vienna, Austria.
collapses the DNA via electrostatic interactions. Of the cationic polymers, PEI is the most popular for gene delivery as DNA/PEI complexes display high transfection efficiency in cell culture and have potential for gene delivery in vivo (5, 6). For tumor specific uptake a ligand such as epidermal growth factor (7), folate (8), or transferrin (9) is conjugated to the cationic polymer. However, in vivo PEI/DNA complexes have been shown to interact with blood components and nontarget cells, strongly reducing their tumor targeting efficiency (10). To overcome this problem a shielding molecule such as the hydrophilic PEG molecule is conjugated to the surface of the DNA complex (11). It is believed that the highly mobile and hydrated PEG strands sterically prevent nonspecific interactions with the biological environment, prolonging the circulation time of the complex in vivo (10). Improved systemic circulation of DNA/PEI complexes by their PEGylation was first described by Ogris et al. (10) with TF-PEI 800 kDa/DNA complexes. PEG was covalently attached to the cationic polymer after DNA polymer condensation (post-PEGylation). PEGylation not only improves systemic tumor targeting but improves solubility of the DNA complexes allowing for formulations to be prepared at the higher therapeutic concentrations without aggregation (10, 12). Furthermore, PEGylation reduces the in vivo toxicity of DNA complexes containing PEI (10, 13).
10.1021/bc0256087 CCC: $25.00 © 2003 American Chemical Society Published on Web 12/05/2002
Stabilized Tumor-Targeted Polyplexes
Bioconjugate Chem., Vol. 14, No. 1, 2003 223 Table 1. Conjugation Ratio of Shielding Conjugates Generated conjugate
ratio (PEI:PEG)
PEI22-PEG20 PEI25-PEG20 PEI22-PEG5 PEI25-PEG5 PEI22-PEG40 PEI25-PEG40 PEI22-ss-PEG5
1/0.9 1/0.9 1/4.4 1/23 1/0.1a, 1/0.3b 1/0.4 1/0.7
a PEI was incubated with PEG succinimide for 1 h. b PEI was incubated with PEG succinimide for 24 h.
Figure 1. A schematic presentation of alternative strategies for the formation of a PEG shielded, ligand-containing DNApolymer complex. Strategy A: DNA is condensed with a polycation (step 1), heterobifunctional PEG is covalently attached to the amino groups of the polycation at the surface (step 2), ligands are covalently attached to the distal end of the PEG chain (step 3). Strategy B: DNA-condensing agent PEI, targeting conjugate ligand-PEG-PEI and shielding conjugate PEGPEI are mixed together (step 1), DNA in buffer is then added and mixed (step 2).
Recently, it was observed that DNA complexes with lower molecular weight PEIs (25 kDa branched, 22 kDa linear) display less systemic toxicity than the PEI 800 kDa complexes (9). Subsequently it was shown that DNA complexes of low molecular weight PEI could also be successfully post-PEGylated. The major drawback of post-PEGylation of DNA complexes is that they require an additional sequential synthesis step which is time-consuming; furthermore, the degree of surface PEGylation is not well defined. Another strategy for the PEGylation of DNA complexes is to synthesize a copolymer of the condensing and the shielding agent prior to the mixing of DNA, for instance, polylysine-PEG (8, 14-16) and PEI-PEG (17-19) copolymers. However, the major limitation of these copolymers is that the hydrophilic part appears to hinder proper DNA condensation and particle formation (14, 17). Work with an alternative shielding agent, transferrin, showed that although Tf-PEI (22 and 25 kDa) conjugates were not able to condense DNA either, the inclusion of extra unmodified PEI25 in the complex enabled efficient DNA condensation and particle formation (9, 20). Since post-PEGylation was not required for these complexes, they could be quickly and easily generated by the flash mixing of three components, Tf-PEI, unmodified linear PEI22, and DNA. In this work we present novel tumor-targeting PEG shielded DNA complexes which can be formed more quickly and easily generated than post-PEGylation strategies (7) (Figure 1). Furthermore these new DNA complexes containing the PEI-PEG shielding conjugate were shown to have tumor-targeting activity after freezethawing. MATERIALS AND METHODS
Reagents and Assays. Branched PEI with an average molecular weight of 25 kDa (PEI25) was obtained from Sigma-Aldrich (Vienna, Austria). Linear PEI with an average molecular weight of 22 kDa (PEI22) is available from Euromedex (Exgen 500, Euromedex, Souffelweyersheim, France). For complex preparation, PEI was used as a 1 mg/mL working solution, neutralized with HCl. Liquid chromatography of conjugates was performed with a Merck-Hitachi L-6220 pump, with a L-4500A UV-vis diode array detector. PEI content of conjugate fractions was determined spectrophotometrically by ninhydrin assay at 570 nm (21) and DNA mobility shift assay by
agarose gel electrophoresis. PEG derivatives were obtained from Shearwater Polymers (Birmingham, AL). The modification of PEI with PEG (molar ratio of PEI/ PEG) in conjugates was determined by proton NMR spectrometry (400 MHz, Bruker, Germany). Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was purchased from Fluka (Buchs, Switzerland) and 2-iminothiolane from Sigma (Vienna, Austria). The amount of dithiopyridine linkers in modified PEI was determined after reduction of an aliquot with dithiothreitol (DTT) followed by absorption measurement of released pyridine-2-thione at 340 nm. The amount of free mercapto groups was determined by Ellman assay at 412 nm (22) using 5,5′dithiobis(2-nitrobenzoic acid) (Sigma, Vienna, Austria). Transferrin (iron-free) was obtained from Biotest (Dreieich, Germany), cell culture media, antibiotics, fetal calf serum (FCS) from Life Technologies (Gaithersburg, MD). Plasmid pCMVLuc (Photinus pyralis luciferase under control of the CMV enhancer/promoter) described in Plank et al.(23) was purified with the EndoFree Plasmid Kit from Qiagen (Hilden, Germany). Plasmid pGSmuTNFR coding for murine TNFR was purified by ELIM Biopharmaceuticals (San Francisco, CA). Endotoxin (LPS) levels were 1000 (aggregates)
aggregates
214 ( 22
+5.6 ( 1.5
PEG-PEI shielded
a
271 ( 34a 1250 ( 118b
+2.8 ( 0.2a +2.3 ( 0.1b
Glucose was added after complex formation and before freezing. b Glucose was added after thawing.
its high surface charge of +31 mV (data not shown) which will facilitate its binding to the cell surface by electrostatic attraction (28). The third control “3” consists of DNA, PEI, and the shielding conjugate PEI22-PEG40, analogous to formulation “a” but lacking the targeting conjugate. The inclusion of the shielding conjugate in the complex reduced the surface charge of the complex to +10 mV (data not shown). The transfection efficiency of this complex was about 100-fold lower than the analogous complexes containing Tf ligand (Figure 3C, compare with formulation “a”). The lower transfection efficiency in comparison to the other control complexes can be attributed to the lower surface charge of the complex, resulting in reduced electrostatic interactions with the cell surface. The higher transfection rate of the transferrin targeting DNA complexes “a” indicates transferrin receptor-mediated internalization of the complexes in K562 cells, which is consistent with our previous work (26, 27, 29). Freeze-Thawing of DNA Complexes for in Vivo Application. It would be of great convenience to store DNA complexes frozen and thaw them prior to their in vivo application. Since previous work demonstrated a strong correlation between the maintenance of particle size/zeta potential and retention of transfection rates (30), the biophysical parameters (particle size and zeta potential) of complexes before and after freeze-thawing was first determined. To determine whether the inclusion of PEG conjugates in the complex affords greater freezethawing stability, we compared Tf-targeting DNA complexes with and without PEG conjugates. The ratio of the components in the complexes is shown in Table 2. To deliver therapeutic amounts of DNA to tumorbearing mice, a higher concentration of DNA (200 µg/ mL) is required. The physical parameters of the complexes were determined with DNA at the higher in vivo concentration of 200 µg/mL, with an N/P of 6. However complexes containing such high concentrations of DNA have been shown to have a higher tendency to aggregate (30). To prevent aggregation of DNA particles, a greater amount (20-30%) of the shielding conjugate in the complexes is required to maintain particles sizes similar to those in the earlier in vitro transfection studies (Figure 4A). In these biophysical studies, PEGylated complexes contained 20% shielding conjugate (Table 2). Complexes were prepared in 20 mM HEPES, pH 7.1, in the absence of NaCl, as previously it was shown that NaCl is a driving force for particle aggregation (30, 31). Furthermore, previous work showed that sugars (sucrose, trehalose, or maltose) were required to maintain size and transfection efficiency of DNA/polymer complexes after freeze-thawing (32). Therefore to minimize aggregation, glucose was added to the complexes after complex formation and before freezing at 5% (w/v) to obtain iso-osmolarity. The effect of adding glucose only after thawing was also investigated as indicated in Table 2. Both PEGylated and non-PEGylated complexes freshly prepared had similar particle size and a surface charge
Figure 4. Transfection efficiency of optimized DNA complexes prepared with DNA at a higher concentration for in vivo application. Complexes were prepared with DNA at 200 µg/mL, N/P ratio of 6.0, in 20 mM HEPES pH 7.1, snap frozen in liquid nitrogen and stored at -20 °C. DNA complexes were thawed in glucose (5% w/v, final concentration). (A) In vitro transfection: the complexes were diluted after thawing with 20 mM HEPES, pH 7.1, to a DNA concentration of 10 µg/mL. Luciferase activity of K562 cells transfected with 5 µg pCMVL DNA per 5 × 105 cells represents the mean ( SD of four to five experiments. (B) Biodistribution of reporter gene expression after systemic gene delivery. The DNA complexes (pCMVL, 50 µg per mouse) after thawing in glucose were injected into the tail vein of Neuro2a tumor-bearing A/J mice. The control complexes (Tf-PEI/PEI25, N/P of 4.8) were freshly prepared as previously described. The biodistribution of gene expression was determined after 24 h by luciferase activity (mean ( SD, n ) 6 animals per group).
near neutral (Table 2). After freeze-thawing of these complexes, the particle size and zeta potential of the PEGylated complex was comparable to the fresh complex, while the non PEGylated complexes grew in size (Table 2). These results suggest that PEGylation can not only stabilize DNA/polymer particles in solution but also prevent aggregation upon the freeze-thawing process. Similarly, it was shown that a DNA-peptide condensing agent when PEGylated was an efficient lyoprotectant, with respect to maintaining particle size (33).
Stabilized Tumor-Targeted Polyplexes
Interestingly, larger but stable PEGylated particles could be generated when glucose was only added after thawing (Table 2). Larger Tf-PEI/DNA complexes (∼1 µm) have previously been shown to have higher transfection efficiencies (30). Therefore these large, stable DNA complexes were used to deliver plasmid DNA systemically to mice. Formulation of DNA Complexes for in Vivo Application. DNA complexes were formed with the higher concentration of DNA (200 µg/mL) and shielding conjugate (20-30%). Complexes were snap frozen and stored, and glucose was added to the complexes after they were thawed, as described in Table 2. Figure 4A shows the physical characteristics and in vitro transfection efficiencies of the three optimized formulations generated at the higher DNA concentration of 200 µg/mL. The particles which we have chosen for further evaluation have a diameter of around 1 µm, slightly larger in size than those prepared with lower amounts of DNA (compare with Figure 3). Importantly for in vivo application, the increased amount of the shielding conjugate in the complex maintains the zeta potential at near neutral. The in vitro transfection efficiencies of the optimized formulations are all much higher than the control DNA/PEI22 complex which aggregated upon the freeze/thaw process. Furthermore, there was no significant difference in the transfection efficiency of all three optimized formulations. To test these optimized formulations in vivo (Figure 4B), each formulation was thawed, glucose added, and the formulation injected into the tail vein of A/J Neuro2a neuroblastoma tumor-bearing mice. The major internal tissues were recovered after 1 day. The control DNA/TfPEI/PEI25 complexes had to be prepared fresh before application as previously described (9). Figure 4B shows the luciferase protein expressed in the respective tissues. For all formulations, the highest expression was observed in the tumor cells. The highest expression in the tumor tissue was observed for PEI22-SS-PEG5 shielded formulation, with an expression level almost 1 log unit higher than the other formulations tested. The luciferase was found to be also expressed at low but significant levels in the liver, followed by the lung. The gene expression in the kidney for the PEI22-PEG20 formulation was due to expression in only 1 mouse out of 6 and in no other treatment group. It is therefore believed that this single observation is animal-specific (possibly due to a dysfunctional kidney) and not formulation-specific. Systemic Administration of a Therapeutic Gene (TNFr). Previously we demonstrated that freshly prepared transferrin-shielded DNA complexes mediate the expression of the highly potent and toxic protein tumor necrosis factor (TNFR) in distant tumors, resulting in therapeutic effects in several murine tumor models (3, 34). On the basis of the previous luciferase experiments, we now selected the cryoconserved formulation shielded by PEI22-SS-PEG5 and tested for its ability to target the TNFR gene expression in distant tumors after systemic application. This was assessed by the reduction in growth and necrosis of tumors in three different murine tumor models, Neuro2a, M-3, and B16F10 (Figure 5). Complexes containing the plasmid pGSmuTNFR coding for murine TNFR were formed in 20 mM HEPES pH 7.1 and stored at -20 °C. Prior to injection, DNA formulations were thawed, glucose was added, and the formulation was injected into the tail vein of mice, between 5 and 7 times at the intervals indicated in Figure 5, and tumor sizes of the animals were recorded. Administration of the formulation was well tolerated by the mice; out of the 174 applications no sign of toxicity was
Bioconjugate Chem., Vol. 14, No. 1, 2003 229
Figure 5. Tumor growth after the systemic application of TfPEG-PEI22/PEI22/PEI22ssPEG5/DNA (pGSmuTNFR) to (A) Neuro2a tumor-bearing A/J mice, (B) M-3 tumor-bearing DBA/ 2mice, or (C) B16F10 tumor-bearing C57bl6 mice. Complexes were prepared in 20 mM HEPES, pH 7.1, using a molar PEI ratio of 1:6:3. DNA complexes were snap frozen in liquid nitrogen and stored at -20 °C. At indicated time points (see arrows), the DNA complex was thawed in glucose (5% w/v final concentration), and the complex (50 µg DNA per mouse) was injected into the tail vein of the mouse (open circles). Control mice were injected with 0.9% (w/v) NaCl (closed circles). Tumor sizes were monitored, mean ( SD are presented; n ) 10 animals in Neuro2a control group, n ) 8 animals per group in all other control and treatment groups. Number of treatment animals with tumor necrosis is indicated in brackets. (*) p < 0.1, (**) p < 0.05, compared to control.
observed. Retardation in tumor development was observed in all three tumor models (Figure 5) accompanied with pronounced tumor necrosis. This is in agreement with transferrin-shielded DNA complexes which localized TNFR activity to the tumor without systemic toxicity (3). Of the models tested, the Neuro2a model showed the strongest response with regard to tumor necrosis with strong necrosis in six of the eight mice treated with the plasmid coding for TNFR, while for the control, two out of the 10 mice only showed weak central necrosis. For both M-3 and B16F10 tumor models, three of the eight mice showed tumor necrosis, while for the controls, no change in tumor growth was observed. Current literature suggests that biodistribution of therapeutic particles following intravenous administration is modulated by the size of the particle and surface charge. A hydrophilic and uncharged surface on the particle reduces their uptake by the phagocytic cells of the RES and aggregation, significantly prolonging circulation time. For targeting DNA complexes to cancer cells, particles should be small enough to pass through the fenestrations in the tumor vasculature (35). These two parameters, particle size and surface hydrophilicity, can easily be modulated by this new streamlined system for generating PEGylated DNA particles. For instance, particle size and surface charge was reduced by increasing the amount of the shielding conjugate in the complex (Figure 2). The in vivo studies (Figure 4B) show that the PEGylated DNA complexes encoding TNFR were effective
230 Bioconjugate Chem., Vol. 14, No. 1, 2003
in tumor necrosis in three different murine tumor models (Figure 5). Despite these promising in vivo results, the tumors were only rarely regressed and there was a variable response in the different tumor models tested. The variable response of the different tumor models to the formulation may be a reflection of the different tumor types having varying leakiness (35). Future studies will use this system to optimize size and surface charge of the transferrin-targeted formulations for targeting different tumors in vivo. CONCLUSION
This work describes a simple and quick method for the formation of PEG shielded transferrin-targeting PEI/ DNA complexes. We demonstrate that the PEG shielded DNA/polymer vector can maintain its size/charge and transfection efficiency after freeze-thawing. For the first time we show that freeze-thawed polyplex vectors have systemic targeting activity toward disseminated tumors. Furthermore, the ratios of the shielding and targeting components in the complex can easily be varied, producing well defined particles with varying size and surface charge. This will enable such biophysical parameters to be easily investigated for their ability to target tumors, leading to DNA vectors which are tailor-made for their application. The simplicity of their preparation and storage in frozen form is a major step forward in the development of nonviral vectors as pharmaceutical products. ACKNOWLEDGMENT
We are grateful to Alexandra Schreiber and Sandra Fandl for help in the animal experiments. LITERATURE CITED (1) Ogris, M., and Wagner, E. (2002) Targeting tumors with nonviral gene delivery systems. Drug Discovery Today 15, 479-485. (2) Felgner, P. L., Barenholz, Y., Behr, J. P., Cheng, S. H., Cullis, P., Huang, L., Jessee, J. A., Seymour, L., Szoka, F., Thierry, A. R., Wagner, E., and Wu, G. (1997) Nomenclature for synthetic gene delivery systems [editorial]. Hum. Gene Ther. 8, 511-512. (3) Kircheis, R, Wightman, L., Kursa, M., Ostermann E, and Wagner, E. (2002) Tumor-targeted gene delivery: an attractive strategy to use highly active effector molecules in cancer treatment. Gene Ther. 9, 731-735. (4) Anchordoquy, T. J., Dean Allison, S., Molina, M., Girouard, L. G., and Carson, T. K. (2001) Physical stabilization of DNAbased therapeutics. Drug Discovery Today 6, 463-470. (5) Boussif, O., Lezoualc’h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. U.S.A. 92, 7297-7301. (6) Ferrari, S., Moro, E., Pettenazzo, A., Behr, J. P., Zacchello, F., and Scarpa, M. (1997) ExGen 500 is an efficient vector for gene delivery to lung epithelial cells in vitro and in vivo. Gene Ther. 4, 1100-1106. (7) Blessing, T, Kursa, M., Holzhauser, R, Kircheis, R, and Wagner, E. (2001) Different strategies for formulation of PEGylated EGF-conjugated PEI/DNA complexes for targeted gene delivery. Bioconjugate Chem. 12, 529-537. (8) Leamon, C. P., Weigl, D., and Hendren, R. W., (1999) Folate copolymer-mediated transfection of cultured cells. Bioconjugate Chem. 10, 947-957. (9) Kircheis, R., Wightman, L., Schreiber, A., Robitza, B., Rossler, V., Kursa, M., and Wagner, E., (2001) Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application. Gene Ther. 8, 28-40.
Kursa et al. (10) Ogris, M, Brunner, S., Schuller, S., Kircheis, R., and Wagner, E. (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6, 595-605. (11) Oupicky, D., Ogris, M., and Seymour, L., (2002) Development of long-circulating polyelectrolyte complexes for systemic delivery of genes. J. Drug Targeting 10, 93-98. (12) Kichler, A., Chillo´n, M., Leborgne, C., Danos, O., and Frisch, B., (2002) Intranasal gene delivery with a polyethylenimine-PEG conjugate. J. Controlled Release 379-388. (13) Kircheis, R, Schuller, S., Brunner, S., Orgris, M., Heider, K. H., Zauner, W., and Wagner, E. (1999) Polycation-based DNA complexes for tumour targeted gene delivery in vivo. J. Gene Med. 1, 111-120. (14) Wolfert, M. A., Schacht, E. H., Toncheva, V., Ulbrich, K., Nazarova, O., Seymour, L. W., Dash, P. R., and Oupicky, D. (1996) Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block copolymers. Hum. Gene Ther. 7, 2123-2133. (15) Katayose, S. and Kataoka, K. (1997) Water Soluble Polyion Complex Associates of DNA and Poly(ethylene glycol) Poly(L-lysine) Block Copolymer. Bioconjugate Chem. 8 (5), 702707. (16) Toncheva, V., Wolfert, M. A., Dash, P. R., Oupicky, D., Ulbrich, K., Seymour, L. W., and Schacht, E. H. (1998) Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers. Biochim. Biophys. Acta 1380, 354-368. (17) Erbacher, P, Bettinger, T., Belguise, Valladier P., Zou, S., Coll, J. L., Behr, J. B., and Remy, J. S. (1999) Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI). J. Gene Med. 1, 210-222. (18) Nguyen, H. K., Lemieux, P., Vinogradov, S. V., Gebhart, C. L., Gue´rin, N., Paradis, G., Bronich, T. K., Alakhov, V. Y., and Kabanov, A. V. (2000) Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents. Gene Ther. 7, 126-138. (19) Woodle, M. C., Scaria, P., Ganesh, S., Subramanian, K., Titmas, R., Cheng, C., Yang, J., Pan, Y., Weng, K., Gu, C., and Torkelson, S. (2001) Sterically stabilized polyplex: ligandmediated activity. J. Controlled Release 74, 309-311. (20) Ogris, M, Steinlein, P., Carotta, S., Brunner, S., and Wagner, E. (2001) DNA/polyethylenimine transfection particles: influence of ligands, polymer size, and PEGylation on internalization and gene expression. AAPS PharmSci. 3, E21. (21) Sarin, V. K., Kent, S. B., Tam, J. P., and Merrifield, R. B. (1981) Quantitative monitoring of solid-phase peptide synthesis by the ninhydrin reaction. Anal. Biochem. 117, 147157. (22) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979) Ellman’s reagent: 5,5′-dithiobis(2-nitrobenzoic acid)- -a reexamination. Anal. Biochem. 94, 75-81. (23) Plank, C., Zatloukal, K., Cotten, M., Mechtler, K., and Wagner, E. (1992) Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chem. 3, 533-539. (24) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. NSuccinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. Biochem. J. 173, 723-737. (25) Kircheis, R, Wightman, L., and Wagner, E. (2001) Design and gene delivery activity of modified polyethylenimines. Adv. Drug Delivery Rev. 53, 341-358. (26) Kircheis, R., Kichler, A., Wallner, G., Kursa, M., Ogris, M., Felzmann, T., Buchberger, M., and Wagner, E. (1997) Coupling of cell-binding ligands to polyethylenimine for targeted gene transfer. Gene Ther. 4, 409-418. (27) Cotten, M., Langle, Rouault, Kirlappos, H., Wagner, E., Mechtler, K., Zenke, M., Beug, H., and Birnstiel, M. L. (1990) Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc. Natl. Acad. Sci. U.S.A. 87, 4033-4037.
Stabilized Tumor-Targeted Polyplexes (28) Mislick, K. A. and Baldeschwieler, J. D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354. (29) Wagner, E., Curiel, D., and Cotten, M. (1994) Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis. Adv. Drug Delivery Rev. 14, 113-136. (30) Ogris, M., Steinlein, P., Kursa, M., Mechtler, K., Kircheis, R., and Wagner, E. (1998) The size of DNA/Transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther. 5, 1425-1433. (31) Wightman, L., Kircheis, R., Ro¨ssler, V., Carotta, S., Ruzicka, R., Kursa, M., and Wagner, E. (2001) Different behavior of branched and linear polyethlyenimine for gene delivery in vitro and in vivo. J. Gene Med. 3, 362-372. (32) Cherng, J. Y., Wetering P., Talsma, H., Crommelin, D. J., and Hennink, W. E. (1999) Stabilization of polymer-based
Bioconjugate Chem., Vol. 14, No. 1, 2003 231 gene delivery systems. Int. J. Pharm. 183, 25-28. (33) Kwok, K. Y., Adami, R. C., Hester, K. C., Park, Y, Thomas, S., and Rice, K. G, (2000) Strategies for maintaining the particle size of peptide DNA condensates following freezedrying. Int. J. Pharm. 203, 81-88. (34) Kircheis, R, Ostermann, E, Wolschek, M. F., Lichtenberger, C., Magin-Lachmann, C., Wightman, L., Kursa, M., and Wagner, E. (2002) Tumor-targeted gene delivery of tumor necrosis factor-alpha induces tumor necrosis and tumor regression without systemic toxicity. Cancer Gene Ther. 9, 673-680. (35) Hashizume, H, Baluk, P, Morikawa, S., McLean, J. W., Thurston, G., Roberge, S., Jain, R. K., and McDonald, D. M. (2000) Openings between defective endothelial cells explain tumor vessel leakiness. Am. J. Pathol. 156, 1363-1380.
BC0256087
