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Poly(Amidoamine)s as Potential Nonviral Vectors: Ability to Form Interpolyelectrolyte Complexes and to Mediate Transfection in Vitro Simon C. W. Richardson, Nicola G. Pattrick, Y. K. Stella Man, Paolo Ferruti,† and Ruth Duncan* Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, U.K.; and Dipartimento di Chimica Organica e Industriale, Universita di Milano, Via Venezian 21, 20133 Milano, Italy Received April 19, 2001
Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity. PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape. The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro. PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10 500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn ∼8000) were studied. All PAAs retarded the electrophoretic mobility of λ Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM. DNase II inhibition was observed. At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 ( 6.1%), ISA 4 (92.2 ( 11.2%), ISA 22 (69.4 ( 3.7%), and ISA 23 (58.0 ( 10.0%). PAAs demonstrated the ability to mediate pSV β-galactosidase transfection of HepG2 cells. At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE. These properties suggest that PAAs warrant further development as endosomolytic vectors. Introduction There are many potential clinical advantages associated with gene, antisense, and ribozyme therapies,1 but they all share the common challenge of effective delivery. Efficient cellular targeting and also localization to the appropriate subcellular compartment is vital if these new approaches are to be converted into useful medicines. Nonviral vectors typically display very poor transfection efficiency transfecting 1 in 103-105 cells under optimal conditions,2 and this has in part been attributed to their inability to efficiently permeabilize the endosomal membrane,3 thus limiting access to the cytoplasm. Addition of fusogenic peptides to the culture medium can increase transfection efficiency,4-6 and it appears that polymeric transfection agents such as polyethleneimine (PEI) can also facilitate transfection in vitro by swelling within the endosome causing membrane rupture, the so-called “proton sponge hypothesis”.7 We have been developing a new family of polymeric drug carriers (reviewed in refs 8 and 9) and nonviral vectors based on synthetic, linear poly(amidoamine) (PAA) polymers. Unlike many lipidic vectors10-12 and polycationic polymers,13 PAAs can be prepared that are nontoxic in vitro11,14 and in vivo.15 PAAs also do not have the immunogenicity disad* Corresponding author. Address: Centre for Polymer Therapeutics, Welsh School of Pharmacy, Redwood Building, Cardiff University, King Edward VII Avenue, Cardiff CF10 3XF, U.K. Telephone: +44 (0)2920874180. Fax: +44 (0)2920874536. E-mail:
[email protected]. † Universita di Milano.
vantage of viral delivery systems,16 and PAA structures can be synthesized that avoid rapid clearance by the reticuloendothelial system.11 Most importantly, amphoteric PAAs undergo a change in tertiary conformation in response to a drop in pH11,17 over the physiologically relevant pH range of 7.4-5.5.18,19 The conformational change is due to protonation of tertiary amino groups arranged regularly along the polymer backbone and this phenomenon confers the ability to selectively damage biological membranes at low pH.11 Unlike many other polyamines such as PEI and poly(L-lysine) (PLL), protonation of the repeating units along the PAA backbone are independent events, thus providing one with the ability to tailor molecules with very sharp acid base dissociation constants.9,18 The purpose of this study was to use four PAAs (synthesis previously described11 ISA 1 (Mn 6900) and ISA 23 (Mn 10 500) and their 2-(4-hydroxy)phenylethylamine containing analogues ISA 4 and ISA 22 (Mn ∼ 8000) (Table 1) to study those properties most relevant to their future development for gene delivery. A DNA agarose gel retardation assay was used to investigate PAA interpolyelectrolyte complex (IPEC) formation, and PAA:DNA complexes were also visualized using transmission electron microscopy (TEM). The ability of PAAs to protect calf thymus DNA from degradation by lysosomal DNase II was assessed. PAA-mediated transfection was studied using Hep G2 cells and pSV β-galactosidase as a marker gene; transfection efficiency was compared with that of the commercially available vectors LipofectACE and LipofectIN and also PEI.
10.1021/bm010079f CCC: $20.00 © 2001 American Chemical Society Published on Web 06/02/2001
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Table 1. Chemical Characteristics of the PAAs
a M (number-average molecular weight) and M (weight-average molecular weight) were calculated by gel permeation chromatography against n w polyamidoamine standards.11
Materials and Methods Materials. All general reagents were from Sigma (Dorset, U.K.) or BDH (Dorset, U.K.) and were of analytical grade unless otherwise stated. Cell culture plastics were from Corning Costar (Buckinghamshire, U.K.). Poly(L-lysine) Mw 56 500 and dextran 72 000 were from Sigma. PEI 70 000 was from Polysciences (Amherst, MA) and contained a minimal degree of branching. Calf thymus DNA and DNase II (EC 3.1.4.6) were from Sigma (Dorset, U.K.), λ Hind III DNA, pSV-β-Galactosidase, Bam H1, Vsp I, and the β-galactosidase assay kit were from Promega, (Maddison, WI). Escherichia coli DH5a, LB broth and ampicillin, minimal essential media (incorporating Earl’s salts (E-MEM), foetal bovine serum (FBS), L-glutamine, and nonessential amino acids (NEAA) were all from Gibco (Paisley, U.K.). Hep G2 was from (ECCAC, Wiltshire, U.K.). In brief, the PAAs were synthesized and characterized as previously described by hydrogen-transfer polyaddition of
the selected bis(secondary amines) to bis(acrylamides)11 (Table 1). In the case of ISA 22 and ISA 23, the monomers involved were 2-methylpiperazine and 2,2′-bis(acrylamido)acetic acid (BAC), and for ISA 1 and ISA 4, the monomers were a mixture of 2-methylpiperazine and N,N-bis(2-hydroxyethyl)ethylenediamine and 1,4-bis(acryloylpiperazine). Both ISA 4 and ISA 22 also contained ∼ 2.5% 2-(4hydroxy)phenylethylamine-derived units.11 IPEC Formation. PAAs, PEI or dextran (1 mg/mL) were added to λ Hind III DNA (0.1 mg/mL) dissolved in 0.9% (w/v) sterile sodium chloride at the mass ratios stated. Complexes were allowed to form for 3 min without mixing. Electrophoresis used a 0.8% (w/v) agarose gel (50 × TAE: 242 g of Tris base, 57.1 mL of glacial acetic acid, and 100 mL of EDTA pH 8, adjusted to pH 7.2 and brought to a final volume to 1 L with distilled water, autoclave) containing 0.25 µg/mL ethidium bromide.20 In each case 1 µg of DNAequivalent was loaded per well and the samples were subject
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to electrophoresis for 60 min at 100 V. Gels were visualized using a UV transilluminator (SLS) and photographed. The morphology of IPECs was also visualized using a Philips 201 TEM by staining IPECs (prepared as above) with 1% uranyl acetate. Protection against Enzymatic Degradation. The procedure was modified from a previously described method.21 Calf thymus DNA was dissolved in 0.2 N sodium acetate buffer pH 5.5 supplemented with 0.2 N potassium chloride (100 µg/mL) and left for 24 h at 4 °C. Prior to assay, 1 mL of 100 mg/mL DNA or IPEC solution was warmed to 37 °C and then DNase II (EC 3.1.4.6) added to a concentration of 300 U/mL. The incubation was conducted at 37 °C throughout. At time zero, duplicate 500 µL aliquots were removed to serve as reference blanks and further samples were taken over 2-60 min. Perchloric acid (500 µL; 10% v/v; 4 °C) was added to each sample and they were then incubated for 30 min at 4 °C prior to centrifugation at 1200g for 20 min (room temperature). The supernatant was removed and decrease in absorbance (260 nm) used to measure DNA degradation. Transfection Studies. The plasmid pSV-β-galactosidase was transformed into E. coli DH5a grown in LB broth containing ampicillin (50 µg/mL) and then isolated using the cleared lysate method.20 Restriction analysis indicated the successful amplification and isolation of the plasmid which was characterized by restriction analysis using Bam H1 and Vsp I and agarose electrophoresis (again using a 0.8% agarose gel containing 0.25 ethidium bromide (µg/mL). Hep G2 cells were grown in 75 mm2 tissue culture flasks using E-MEM containing 10% FBS and 5 mM L-glutamine and 1% NEAAs. When ∼70% confluence was attained, 1 × 106 cells/well were seeded into six-well tissue culture plates, and after 24 h, the pSV-β-galactosidase transfection assay was initiated. First, sterile vehicle (polymer or lipid) was added to plasmid (1 µg) (to give the appropriate vehicle: DNA ratio), and the volume was adjusted to 50 µL with sterile PBS. This was then added to the cells and subject to gentle aspiration. After 48 h β-galactosidase was assayed using a commercially available kit (Promega E2000) was used to assay for increases in β-galactosidase activity spectrophotometrically. The results were expressed as units of β-galactosidase activity/cell relative to protein concentration assayed using the BCA assay. A statistical analysis was performed using a Student’s t test integrated into Prism 2.0a software (GraphPad, Software Inc., San Diego, CA). Results Figure 1 shows the migration of λ Hind III DNA in the presence of various polymers. The distance migrated and the intensity of ethidium bromide staining indicate the degree of retardation. ISA 1 and ISA 4 (Figure 1a) caused DNA retardation that was greater than that seen with PEI at an equivalent mass ratio (1:1). DNA retardation mediated by ISA 22 and ISA 23 is shown in Figure 1b. The reduction in ethidium bromide staining seen in lanes 2 and 3 relative to lanes 4 and 5 indicates DNA condensation is occurring, but no obvious retardation is observed. It can be seen from Figure 2 that ISA 4 and ISA 22 form discrete toroids of between
Figure 1. DNA retardation during agarose gel electrophoresis of polymer complexes. The gels were loaded at 1 µg of λ Hind III DNA/ well. The key is indicated on the plate. At the higher ratios an excess of polymer was used. Panel a shows ISA 4, ISA 1, and the controls PEI and dextran. Panel b shows ISA 22 and ISA 23.
80 and 150 nm following mixing with plasmid. The size and morphology of the naked plasmid is shown for comparison. Similar toroids were seen when the plasmid was mixed with PLL, whereas in contrast the LipofectACE:DNA complexes had a very disordered morphology. Significant DNase II protection occurred after IPEC formation with all PAAs (Table 2). Figure 3, parts a and b,
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Figure 2. Transmission electron microscopy showing morphology of the vector-DNA complexes. In each case complexes were formed at a 10-fold vector excess. Key: (a) LipofectACE (size bar 67 nm), (b) poly(L-lysine) (size bar 67 nm), (c) ISA 22 (size bar 45.5 nm), (d) ISA 4 (size bar 140 nm), and (e) plasmid (size bar 67 nm). Table 2. Effect of PAAs, Dextran, and Poly(ethylenimine) on the Degradation of Calf Thymus DNA by DNase II mass ratioa polymer
100:1
10:1
1:1
0.1:1
ISA 1 ISA 4 ISA 22 ISA 23 PEI dextran
ND ND 79.5 ( 0.3 74.5 ( 7.3 ND 12.7 ( 6.6
89.6 ( 6.1 92.2 ( 11.2 69.4 ( 3.7 58.0 ( 10.0 100.0 17.5 ( 2.0
86.5 ( 1.9 87.2 ( 3.3 12.7 ( 2.7 34.5 ( 0.7 100.0 ND
ND ND ND ND 49.9 ( 13.6 ND
a
Results are expressed as % inhibition. ND: Not Determined.
shows typical time courses for degradation of ISA 4 and ISA 22, respectively. Comparison of the extent of protection afforded by the PAAs compared to that seen using dextran or PEI at a 10:1 polymer excess (Figure 3c) gave a rank order: PEI > ISA 1 ) ISA 4 > ISA 22 ) ISA 23 > dextran. Dextran did not protect against degradation to any significant extent and PEI gave 100% protection at polymer: DNA ratios of 10:1 and 1:1. Even at the lower ratio of 0.1:1 the PEI IPEC displayed 49.9 ( 13.6% protection after the 60 min incubation with DNase II. To study PAA-mediated transfection, polymers and the various control vectors were used in combination pSV-βgalactosidase and HEPG2 cells. First the effect of vector: DNA ratio was studied (Figure 4a) using LipofectACE as a reference control. The greatest level of β-galactosidase expression was mediated by ISA 23 at 1:9 ratio with excess polymer. ISA 23 showed a higher level of β-galactosidase
Figure 3. Polymer-mediated DNA protection against DNase II degradation. Key: (a) DNA alone (2s2), ISA 4:DNA (10:1) (9s9), and ISA 4:DNA (1:1) (bsb); (b) DNA alone (2s2), ISA 22:DNA (10: 1) (9s9), and ISA 22:DNA (1:1) (bsb); (c) dextran:DNA (10:1) (2s 2), PEI:DNA (10:1) (0 0), ISA 4:DNA (10:1) (9s9), and ISA 22: DNA (10:1) (4s4). In each case the ratios given are mass ratios.
expression than ISA 22 and also the LipofectACE control at every ratio with the exception of the 10:1 ratio. To allow correlation with known nonviral vectors the transfection efficiency was compared at a 5:1 vector excess (Figure 4b). ISA 23 resulted in a β-galactosidase expression that was equivalent to that seen using PEI and LipofectIN. Both ISA 22 and ISA 23 showed significantly higher expressions than those seen using LipofectACE. Although ISA 1 and ISA 4 did promote transfection, neither was significantly more effective than LipofectACE at the 5:1 ratio.
Poly(amidoamine)s for Gene Delivery
Figure 4. Transfection of Hep G2 cells using pSV-β-galactosidase in the presence of various vectors. Panel a shows the β-galactosidase expression after incubation with square with left-vertical lines ISA 23, ISA 22 (0) and LipofectACE (9) complexes at mass ratios between 1:1 and 1:10 using excess vector. In panel b, the β-galactosidase expressions are compared after exposure of HEP G2 cells to plasmid alone, LipofectIN, LipofectACE, ISA 1, ISA 4, ISA 22, ISA 23, and PEI, and without plasmid. A mass ratio of 5:1 (excess vector) was used throughout.
Discussion Immunogenicity is a major problem facing routine use of viral vectors for repeated gene delivery;19 therefore, rational design of a synthetic polymer-based viral mimetic may provide an intracytoplasmic delivery system lacking immunogenicity but containing all the elements needed to render the system suitable for routine clinical use. It is noteworthy that other synthetic polymers, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, have recently entered clinical trials as anticancer conjugates without signs of immunogenicity.22 The formation of soluble IPECs23 and the polycationmediated condensation of DNA (forming (DNA) are well documented,23,24 and this process is driven by the cooperative interaction of protonated amines within the polymer coil and the anionic nonbridging oxygen integral to the plasmid. The amino groups of all the PAAs studied here are partially ionized at pH 7.4.9 In addition ISA 22 and ISA 23 display amphoteric properties because they carry also a carboxyl
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group which is fully ionized at the relevant physiological pHs (pH 5-7.4).18 It was surprising that ISA 1 and ISA 4 formed IPECs that showed greater retardation than those formed by PEI at an equivalent mass ratio (1:1) (Figure 1), but this may be due to the general chain stiffness of PAAs and also to the fact the charged amino groups are an integral part of the polymer backbone. Interpretation of the lack of obvious gel retardation induced by ISA 22 and ISA 23 (Figure 1), in spite of complex formation (Figure 2), is difficult. It may be due to their amphoteric nature, leaving an excess of negative charges, combined with the remarkable chain stiffness reported for ISA 23.18 The degree of protonation associated with PAAs at specific pHs has been described.18 however, it has been shown previously that assessing the efficiency of interpolyelectrolyte coil interactions and polycation-mediated DNA condensation only from the point of view of charge stoichiometry can be misleading.11,25 All the PAAs demonstrated the ability to protect DNA from the nuclease DNase II (Figure 3 and Table 2), and protection was probably due to steric hindrance.23 Although the protective effect of PEI was much greater than seen for the PAAs, PEI has a relatively higher charge density per monomer and thus might be expected to form tighter complexes. However, this protective effect can only be advantageous if the IPEC can subsequently disassociate in the cytoplasm to liberate its payload. PAAs were able to promote transfection of HEPG2 cells by pSV-β-galactosidase (Figure 4). Interestingly, ISA 23 was equi-effective when compared with PEI and LipofectIN, and both ISA 22 and ISA 23 were more effective than LipofectACE. It is often stated that polymeric transfection agents are limited by endosomal exit3.This is not usually so for lipidic delivery systems. Addition of fusogenic peptides in vitro has been used to enhance polymer-mediated transfection by eliminating this potential bottleneck.26 We believe that when protonated at pH 6.5 PAAs inherently destabilize endosomal membranes and this would be consistent with the pH-dependent breakage of model membranes seen previously, which was particularly evident for ISA 23.11,18 Also this would be consistent with the well-known mechanisms of polycation-mediated bilayer destabilization seen using polymers such as PLL.27,28 However, it cannot be discounted that PAAs also display intraendosomal swelling as reported for PEI7 following endocytic capture which would also induce membrane damage. Generally, for an IPEC to remain stable and mediate gene transfer, it has been suggested that a polymer-DNA complex must maintain a net cationic charge. However, this positive charge is normally associated with both toxicity29,30 and a hepatotropic body distribution.31 As both cationic and anionic macromolecules are predisposed to liver capture, to avoid rapid liver clearance (i.e., if the therapeutic target is not the liver) the polymer or IPEC should be neutral or zwitterionic at pH 7.4.31,32 Although the purely cationic ISA 4 is hepatotropic, it was shown that radiolabeled ISA 22, which is amphoteric, demonstrates “stealthlike” properties, and this polymer can be directed preferentially to solid tumor tissue due to its enhanced vascular permeability.11 Unlike other polycations including PLL and PEI (IC50 ∼ 10 µg/mL)30 and
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the amine-terminated PAMAM dendrimers,33 PAAs can be synthesized to show minimal toxicity.11,14 ISA 1 and 4 have IC50 values of ∼3 mg/mL and ISA 22 and 23 have IC50 values of 4-5 mg/mL, respectively, against B16F10 cells.11 Recent observations that PAAs enter the endocytic pathway and rupture intracellular membranes after intravenous administration34 and also can promote the delivery of nonpenetrating toxins in vitro35 suggests that PAAs may have the potential to delivery a range of macromolecular therapeutics. Acknowledgment. S.C.W.R. and R.D. would like to thank the Academy of Science in Mainz, Germany, and the MRC and the BBSRC for funding their work. N.G.P. is supported by a BBSRC studentship and P.F. acknowledges support of the Italian Ministry of the University and Technological Development (MURST). References and Notes (1) Hersh, E. M.; Stopeck, A. T. In Self-Assembling Complexes for Gene DeliVery from Laboratory to Clinical Trial; Kabanov, A. V., Felgner, P. L., Seymour, L. W., Eds.; Wiley: New York, 1998; pp 421-436. (2) Graham, F. L.; Van der Eb., A. J. Virology 1973, 52, 456-467. (3) Kichler, A.; Mechtler, K.; Behr, J.-P.; Wagner, E. Bioconjugate Chem. 1997, 8, 213-221. (4) Lozier, J. N.; Thompson, A. R.; Hu, P. C.; Read, M.; Brinkhaus, K. M.; High, K. A.; Curiel, D. T. Hum. Gene Ther. 1994, 5, 313-322. (5) Wagner, E.; Plank, C.; Zatloukal, K.; Cotton, M.; Birnstiel, M. L. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7934-7938. (6) Wagner, E.; Zatloukal, K.; Cotton, M.; Kirlappos, H.; Mechtler, K.; Curiel, D. T.; Burnstiel, M. L. Proc. Natl. Acad. Sci. U.S.A.. 1992, 89, 6099-6103. (7) Boussif, O.; Lezoualch, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J.-P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 7297-7310. (8) Ferruti, P. In Polymeric Materials Encyclopedia; Salamone; J. C., Ed.; CRC Press Inc.: Boca Raton, FL, 1996; Vol. 5, pp 3334-3359. (9) Ferruti, P.; Duncan, R.; Richardson, S. In Targeting of Drugs 6: Strategies for Stealth Therapeutic Systems; Gregoriadis, G., McCormack, B., Eds.; Plenum Publishing Corp.: New York, 1998; pp 207224. (10) Bally, M. B.; Harvie, P.; Wong, F. M. P.; Kong, S.; Wasan, E. K.; Reimer, D. L. AdV. Drug Del. ReV. 1999, 38, 291-315. (11) Richardson, S.; Ferruti, P.; Duncan, R. J. Drug Targeting 1999, 6, 391-404. (12) Smith, J.; Zhang, Y.; Niven, R. AdV. Drug Del. ReV. 1997, 26, 135150.
Richardson et al. (13) Godbey, W. T.; Wu, K. K.; Mikos, A. G. J. Controlled Release 1999, 60, 149-160. (14) Ranucci, E.; Spagnoli, G.; Ferruti, P.; Sgouras, D.; Duncan, R. J. Biomater. Sci.: Polym. Ed. 1991, 2, 303-315. (15) Ferruti, P.; Ranucci, E.; Trotta, F.; Gianasi, E.; Evagorou, E. G.; Wasil, M.; Wilson, G.; Duncan, R. Macromol. Chem. Phys. 1999, 200, 1644-1654. (16) Gorecki, D. C. Emerg. Drugs 1999, 4, 247-261. (17) Duncan, R.; Ferruti, P.; Sgouras, D.; Tubokumetzger, A.; Ranucci, E.; Bignotti, F. J. Drug Targeting 1994, 2, 341-347. (18) Ferruti, P.; Manzoni, S.; Richardson, S. C. W.; Duncan, R.; Pattrick, N. G.; Meridichi, R.; Casolaro, M. Macromolecules 2000, 33, 77937800. (19) Sandhu, J. S.; Keating, A.; Hozumi, N. Crit. ReV. Biotechnol. 1997, 17, 307-326. (20) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbour Press: New York, 1986; sections 6.3-6.15. (21) Barrett, A. J.; Heath, M. F. In Lysosomes: A Laboratory Handbook, 2nd ed.; Dingle, J. T., Ed.; North-Holland Publishing Co.: Amsterdam, New York, and Oxford, England, 1977; pp 19-147. (22) Vasey, P.; et al. Clin. Cancer Res. 1945, 5, 83-94. (23) Kabanov, A. V.; Szoka, F. C., Jr.; Seymour, L. W. In Self-assembling complexes for gene deliVery from laboratory to clinical trial; Kabanov, A. V., Felgner, P. L., Seymour, L. W., Eds.; Wiley: New York, 1998; pp 197-218. (24) Manning, G. S. Biopolymers 1980, 19, 37-59. (25) Richardson, S. C. W. Characterisation of poly(amidoamine)s and chitosan as potential intracytoplasmic delivery systems. Ph.D. Thesis, University of London, 1999. (26) Wagner, E. J. Controlled Release 1998, 53, 155-158. (27) Kachalsky, A.; Dannon, D.; Nevo, A.; De Vries, A. Biochim. Biophys. Acta 1959, 33, 120-138. (28) Hartmann, W.; Galla, H.-J. Biochim. Biophys. Acta 1978, 509, 474490. (29) Van de Wetering, P.; Cherng, J.-Y.; Talsma, H.; Hennink, W. E. J. Controlled Release 1997, 49, 59-69. (30) Sgouras, D.; Duncan, R. J. Mater. Sci.: Mater. Med. 1990, 2, 6168. (31) Takakura, Y.; Hashida, M. In Self-assembling complexes for gene deliVery from laboratory to clinical trial; Kabanov, A. V., Felgner, P. L., Seymour, L. W., Eds.; Wiley: New York, 1998; pp 295-306. (32) Yamaoka, T.; Kuroda, M.; Tabata, Y.; Ikada, Y. Int. J. Pharm. 1995, 113, 149-157. (33) Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorenz, K.; Frey, H.; Weener, J. W.; Meijer, E. W.; Paulus, W.; Duncan, R. J. Controlled Release 2000, 65, 113-148. (34) Richardson, S. C. W.; Man, Y. K. S.; Ferruti, P.; Duncan, R. Proc. 26th Int. Symp. Control. Relat. Bioact. Mater. 1999, 26, 434-435. (35) Pattrick, N. G.; Richardson, S. C. W.; Casolaro, M.; Ferruti, P.; Duncan, R. J. Controlled Release 2001, submitted for publication.
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