M E T H O D S I N M O L E C U L A R M E D I C I N E TM Edited by Mark A. Findeis Nonviral Vectors for Gene Therapy Methods and Protocols Humana Press Humana Press Edited by Mark A. Findeis Nonviral Vectors for Gene Therapy Methods and Protocols Synthesis of Polyampholyte Comb-Type Copolymer 1 1 From: Methods in Molecular Medicine, vol. 65: Nonviral Vectors for Gene Therapy Edited by: M. A. Findeis © Humana Press Inc., Totowa, NJ 1 Synthesis of Polyampholyte Comb-Type Copolymers Consisting of Poly( L-lysine) Backbone and Hyaluronic Acid Side Chains for DNA Carrier Atsushi Maruyama and Yoshiyuki Takei 1. Introduction Polycations have been used as nonviral gene carriers because the polycations and DNAs form stable complexes in a noncovalent manner (1–3). The polycations, e.g., poly( L-lysine) (PLL), are reported to be conjugated with several ligands for targeted gene delivery (4–8). The physicochemical properties of the DNA complexes have been described as factors that influence transfection activity (9–11). The authors have reported (12,13) several comb-type copolymers consisting of a PLL backbone and hydrophilic dextran side chains for controling the assembling structure of DNA–copolymer complexes. The dextran chains grafted onto PLL are found to reduce aggregation of the resulting complexes and to increase the solubility of the complexes. Furthermore, the grafting degree of the copolymer affects the DNA conformation in the complex, allowing regulation of DNA compaction. The comb-type copolymers with a higher degree of grafting induce little compaction of DNA and stabilize DNA duplexes and triplexes by shielding the repulsion between phosphate anions of DNA. Moreover, the grafted chains reduce the nonspecific interaction of the PLL backbone with proteins (14). The comb-type copolymers therefore fulfill several requirements for the cell- specific carrier of DNA, if the copolymers are provided with cell-specific ligands. Hyaluronic acid (HA) is an unbranched high-molecular-weight polysaccharide consisting of alternating N-acetyl-`- D-glucosamine and `-D-glucuronic acid resi- dues linked at the 1.3 and 1.4 positions, respectively (15). Liver sinusoidal endothelial cells possess the receptors that recognize and internalize HA (16,17). More than 90% of HAs in the blood stream are known to be taken up and metabo- 2 Maruyama and Takei lized by SECs. The authors are therefore interested in HA as the ligand to deliver the DNA to the SEC. The authors’ recent study (18) shows that the complexes between PLL–HA conjugates and reporter genes were distributed exclusively in SECs, leading to gene expression in vivo. This chapter describes preparation of PLL-graft-HA (PLL-g-HA) comb-type copolymers. For the synthesis of the comb-type copolymers, high-molecular- weight HA was hydrolyzed, then the HAs were covalently coupled with ¡-amino groups of PLL at their reducing end by reductive amination reaction. 2. Materials 2.1. Enzymatic Hydrolysis of HA 1. High-molecular-weight HA (5.9 × 10 2 kDa), obtained as its sodium salt (sodium hyaluronate), was a gift from Denki Kagaku Kogyo (Tokyo, Japan). 2. Bovine testicular hyaluronidase (EC 3.2.1.35; Type I-S, Sigma, St. Louis, MO). 3. Syringe filters, 0.45 µm (New Steradisc 25, Kurabo, Osaka, Japan). 2.2. Synthesis of PLL- g -HA Comb-Type Copolymers 1. PLL, obtained as its chloride or bromide salt, was purchased from Peptide Institute (Osaka, Japan). 2. Sodium borate buffer-NaCl: 0.1 M, pH 8.5, 0.4–1 M NaCl. 3. Sodium cyanoborohydride (NaBH 3 CN). 4. NaCl solution (0.5 M). 5. Dialysis membrane (Spectra/Por 7, mol wt cut-off 25,000). 6. Distilled water. 2.3. Size Exclusion Chromatography (SEC)–Multiangle Laser Light Scattering (MALLS) Apparatus 1. Chromatography pumping system operating at 1.0 mL/min. 2. Size exclusion chromatography column(s). 3. NaNO 3 (0.1 M) containing 5 mM sodium phosphate buffer, pH 8.0. 4. Na 2 SO 4 (0.2 M) containing 5 mM sodium phosphate buffer, pH 8.0. 3. Methods 3.1. Enzymatic Hydrolysis of HA 1. Dissolve hyaluronic acid (1 g) in 120 mL water. 2. Add 20 mg bovine testicular hyaluronidase and stir at 50°C. 3. Use a small portion of the reaction to trace HA molecular weight change by SEC–MALLS. 4. When the desired molecular weight of the HA is reached, boil the mixture for 5 min to terminate the reaction. 5. After cooling down to room temperature, filter the mixture through a 0.45-µm filter to remove the denatured enzyme. The resulting HA fragments were obtained by freeze-drying. Synthesis of Polyampholyte Comb-Type Copolymer 3 Figure 1 shows the time course of the HA hydrolysis determined by a SEC– MALLS apparatus (see Subheadings 2.3. and 3.3.). The rate of hydrolysis depends on enzyme activity, so that preliminary experiment on a small scale is recommended to estimate hydrolysis rate before a large-scale reaction. The rate of hydrolysis can be regulated by changing enzyme concentration. For graft copoly- mer synthesis, a molecular weight ranging from 3000 to 10,000 is favorable. Because the hydrolyzed product has a large distribution in molecular weight, it is also recommended to fractionate fragments by dialysis or ultrafiltration. 3.2. Synthesis of PLL- g -HA Comb-Type Copolymers The obtained HA fragments were conjugated to PLL by reductive amination using NaBH 3 CN as a reducing agent (Scheme 1). The reaction proceeded through two steps. First is the Schiff’s base (-CH=N-) formation between a reductive (aldehyde) end of HA and primary amino groups of PLL. Second is reduction of the Schiff’s base to form secondary amino groups (-CH 2 -NH). Although the Schiff’s base is unstable and reversible, its reduced product is an irreversible covalent product. 1. Dissolve the PLL (60–120 mg) in 15 mL sodium borate buffer (0.1 M, pH 8.5) containing 0.4–1 M NaCl. 2. Add the HA fragment (100–300 mg) to the solution. If turbidity or precipitation was observed, increase NaCl concentration. PLL and HA may form an interpolyelectrolyte complex, which is unfavorable for graft copolymer synthesis. The complex formation can be avoided by increasing NaCl concentration. 3. Stir the mixture at 40°C for a few hours for Schiff’s base formation. Fig. 1. Time-course of the hydrolysis of HA by hyaluronidase detected by SEC– MALLS. HA (5.9 × 10 2 kDa; 1 g) was hydrolyzed by hyaluronidase (20 mg) at 50°C. (mL) 4 Maruyama and Takei 4. Add NaBH 3 CN to the mixture and allow to stand at 40°C for 2 d. Approximately 10 molar excess of NaBH 3 CN to HA is recommended. 5. Sample the solution and trace the reaction with SEC–MALLS (see Subheading 3.3. for SEC–MALLS procedure). 6. Purify the mixture by dialysis against 0.5 M NaCl aqueous solution using a Spec- tra/Por 7 membrane (mol wt cut-off = 25,000). 7. Desalt the sample by dialysis against distilled water, the resulting copolymer is obtained by freeze-drying. The resulting copolymer would be precipitated during the dialysis. Figure 2 shows the time-course of the coupling reaction between PLL and HA traced by SEC–MALLS. The reaction can be detected as a decrease in peak area of free HA, increase in peak of the copolymer and in molecular weight of the copolymer. The coupling was almost completed within a few days of incuba- tion. Note that the free HA is almost eliminated after the reaction. Scheme 1. Synthesis of PLL-g-HA comb-type copolymers. Reprinted with permission from ref. 19. Copyright 1998, American Chemical Society. Synthesis of Polyampholyte Comb-Type Copolymer 5 3.3. SEC–MALLS SEC was carried out using a JASCO 880-PU pumping system (Tokyo, Japan) at the flow rate of 1.0 mL/min at 25°C, with Ultrahydrogel series (Japan Waters, Tokyo, Japan) or Shodex OH pack SB-series (Showa Denko, Tokyo, Japan). A suitable combination of mobile phase and columns must be chosen, because poly- electrolytes including HA and PLL are liable to interact with the column packings, leading to delay in elution volume. In such case, molecular weight determination using the calibration curve based on molecular weight standard samples such as polyethyleneglycol and pullulan is not reliable. The choice of the mobile-phase rely on gel permeation chromatography (GPC) columns. It is highly recommended to provide a light-scattering (LS) detector system such as MALLS (Multiangle laser light scattering detector, Dawn-DSP, Wyatt Technology, Santa Barbara, CA). By using a LS detector, a direct estimation of the molecular weight is possible. The mobile phases we used are 0.1 M NaNO 3 for HA fragment analysis and 0.2 M Na 2 SO 4 containing 5 mM sodium phosphate buffer (pH 8.0) for copoly- mer analysis. For typical analysis, 200 µL of each sample was picked up from the reaction mixture and injected into the columns. Eluate was detected by a refractive index (RI) detector (830-RI, JASCO) and a MALLS detector. RI and LS signals were transferred to a computer to calculate number-average and weight-average molecular weight according to the instruction manual (Wyatt Technology) for Dawn-DSP. 3.4. 1 H Nuclear Magnetic Research (NMR) Spectroscopic Analyses Each copolymer was dissolved in D 2 O (Deuterium content: 99.95% Merck, Darmstadt, Germany) containing 0.35 M NaCl. 1 H-NMR spectra (400 MHz) were Fig. 2. Time-course of coupling reaction between PLL and HA detected by SEC– MALLS. 6 Maruyama and Takei obtained by a Varian Unity 400plus spectrometer (Palo Alto, CA), at a probe temperature of 298 K. The chemical shifts are expressed as parts/million using internal HDO molecules (b = 4.7 ppm in D 2 O) as a reference. As shown in Fig. 3, the 1 H-NMR spectra of the comb-type copolymer showed the characteristic signals of both PLL and HA moieties: PLL, b 1.4–1.8 (`, a, b-CH 2 ), 3.0 (¡-CH 2 ), 4.3 (_-CH); HA, b 2.0 (NAc-CH 3 ), 3.3–3.9 (H-2,3,4,5,6), 4.4–4.6 (H-1). From the signal ratio of methyl protons (2.0 ppm) of the N-acetyl groups of the HA-grafts to ¡-methylene protons (3.0 ppm) of the PLL backbone, the content (wt % and grafting-%) of HA in the copolymer was determined. The results of the synthesis of PLL-g-HA comb-type copolymers are summarized in Table 1. Coupling efficiency was more than about 70%. Consequently, the authors have easily prepared the various PLL–HA conjugates with a well-defined comb- type structure by combining enzymatic hydrolysis and the reductive amination. Fig. 3. 1 H NMR spectra of PLL (A), HA (B), and PLL-g-HA (C) in D 2 O. For the PLL-g-HA, D 2 O containing 0.35 M NaCl was used. Reprinted with permission from ref. 19. Copyright 1998, American Chemical Society. Synthesis of Polyampholyte Comb-Type Copolymer 7 7 Table 1 Synthesis of PLL-g-HA Comb-Type Copolymers a In feed Copolymer Coupling PLL HA Mol wt b HA Content c efficiency d Yield Charge Sample M n b /10 4 mg M n b /10 3 mg wt% M n /10 4 M w /M n wt% Grafting-% ratio % % 1 4.2 61.2 2.3 94.3 61 8.5 1.5 50 5.7 0.34 66 67 2 4.2 61.2 2.3 189 76 11 1.4 63 9.4 0.56 55 57 3 7.2 122 3.8 189 61 15 1.4 53 3.8 0.40 73 55 4 7.2 61.2 3.8 189 76 23 1.6 69 7.6 0.83 73 83 5 7.2 61.2 3.8 283 82 31 1.5 77 11 1.3 71 57 6 4.2 61.2 1.6 94.3 61 9.3 1.4 55 9.8 0.49 80 38 7 7.2 122 1.6 94.3 44 12 1.5 38 4.9 0.22 80 51 8 7.2 61.2 1.6 189 76 24 1.4 71 19 1.0 77 85 a Reducing reagent, 0.3 M NaBH 3 CN; reaction temperature, 40°C; reaction time, 56 h (samples 1 and 2), 80 h (samples 3–5), 75 h (samples 6–8); solvent, 0.1 M sodium borate buffer (pH 8.5) containing 0.4 M NaCl (samples 1 and 2) or 1 M NaCl (samples 3–8). b Molecular weight and its distribution (M w /M n ) were determined by SEC–MALLS. c Determined by 1 H-NMR; grafting-% = (mol fraction of the lysine residues grafted with HA) × 100%; charge ratio = [carboxyl group] HA / [amino group] PLL in copolymer. d [HA] copolymer /[HA] in feed × 100%. Reprinted from ref. 19. Copyright 1998, American Chemical Society. 8 Maruyama and Takei References 1. Kabanov, A. V., Astafyeva, I. V., Chikindas, M. L., Rosenblat, G. F., Kiselev, V. I., Severin, E. S., and Kabanov, V. A. (1991) DNA interpolyelectrolyte complexes as a tool for efficient cell transformation. Biopolymers 31, 1437–1443. 2. 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 oligonucle- otide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301. 3. Page, R. L., Butler, S. P., Subramanian, A., Gwazdauskas, F. C., Johnson, J. L., and Velander, W. H. (1995) Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures. Transgenic Res. 4, 353–360. 4. Wu, G. Y. and Wu, C. H. (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J. Biol. Chem. 262, 4429–4432. 5. 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(1998) Characterization of interpolyelectrolyte complexes between double- stranded DNA and polylysine comb-type copolymers having hydrophilic side chains. Bioconjugate Chem. 9, 292–299. 14. Maruyama, A., Ishihara, T., Kim, J. S., Kim, S. W., and Akaike, T. (1997) Nanoparticle DNA carrier with poly(L-lysine) grafted polysaccharide copolymer and poly(D,L-lactic acid). Bioconjugate Chem. 8, 735–742. Synthesis of Polyampholyte Comb-Type Copolymer 9 15. Balazs, E. A., Laurent, T. C., and Jeanloz, R. W. (1986) Nomenclature of hyalu- ronic acid. Biochem. J. 235, 903. 16. Forsberg, N. and Gustafson, S. (1991) Characterization and purification of the hyaluronan-receptor on liver endothelial cells. Biochim. Biophys. Acta 1078, 12–18. 17. Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) Identification of the Ca 2+ -independent endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells using a photoaffinity cross-linking reagent. J. Biol. Chem. 267, 20,451–20,456. 18. 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[...]... structure, we investigated the influence of the peptide chain length on gene transfer ability As a result, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA, From: Methods in Molecular Medicine, vol 65: Nonviral Vectors for Gene Therapy Edited by: M A Findeis © Humana Press Inc., Totowa, NJ 11 12 Niidome and Aoyagi Table 1 Structures of Amphiphilic -Helical Peptides Peptide... sample loading buffer and load onto the gel 4 Perform electrophoresis for 30 min at 100 V 5 Illuminate the elecrophoresced gel on an UV illuminator to show the location of the DNA and complexes (see Fig 3A,B and Note 7) 3.4 Atomic Force Microscopy 1 Prepare the freshly split mica 2 Prepare the polymer solution (G = 4) and DNA solution in water 3 Mix the two solutions and incubate for 30 min at room temperature... Ulbrich, K., Nazarova, O., and Seymour, L W (1996) Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block co-polymers Hum Gene Ther 7, 2123–2133 6 Kataoka, K., Togawa, H., Harada, A., Yasugi, K., Matsumoto, T., and Katayose, S (1996) Spontaneous formation of polyion complex micelles with narrow distribution from antisense oligonucleotide and cationic block copolymer... transfection performance, random copolymers were synthesized Random copolymers of DMAEMA with ethoxytriethylene glycol methacrylate (triEGMA), N-vinylpyrrolidone (NVP), methyl methacrylate (MMA), and methacrylic acid of different molecular weights and compositions (comonomer fraction up to 66 mol%) are able to bind DNA, yielding polyplexes (14,15; and Bos and Hennink, unpublished data) However, for random copolymers... affect pDMAEMAmediated transfection: Circular forms of DNA (supercoiled and open-circular) show higher transfection activity than linear forms (25) Recently, the possibilities and limitations of autoclaving, filtration, and a combination of both methods for sterilization of pDMAEMA-based gene transfer complexes have been assessed (26) Agarose gel electrophoresis and circular dichroism spectroscopy shows... 11 2 Cationic -Helical Peptides for Gene Delivery into Cells Takuro Niidome and Haruhiko Aoyagi 1 Introduction Development of nonviral gene transfer techniques has progressed, particularly the use of several kinds of cationic lipids and cationic polymers such as polylysine derivatives, polyethyleneimines, polyamidoamine dendrimers, and so on, which electrostatically form a complex with the negatively... After incubation for 12 h at 37°C, the medium is replaced with 1 mL fresh medium containing serum and the cells are further incubated for 24 h 6 Harvest of cells and luciferase assays are performed as described in the protocol of PicaGene luminescence kit using a luminometer 7 The protein concentrations of the cell lysates are measured by Bradford assay using bovine serum albumin as a standard The light... which is standardized for total protein content of the cell lysate The measurement of gene transfer efficiency is performed in triplicate To date, the authors have tested gene transfer efficiencies of the peptides into several cell lines, such as COS-7, HeLa, CHO, and HuH-7 cells Figure 1 shows the results for each cell line To compare the efficiencies of the peptides to a commercially available gene transfer... cytotoxicity, and low transfection efficiency, limited their use as in vivo gene carriers (1) Among them, however, dendrimers are still very attractive to many scientists for the design of gene carriers because of their well-defined structure and ease of control of surface functionality Already, both polyamidoamine dendrimer and polyethylenimine dendrimer have been tested for their potential utility and have... polyspermine, and polyethylenimine) (5–8) or liposomes (9) to improve the solubility of complexes with DNA and transfection efficiency This chapter provides the design of a conceptually new hybrid block copolymer which is capable of polyionic complex formation with plasmid DNA via supramolecular self-assembly Linear PEG was coupled to the globular From: Methods in Molecular Medicine, vol 65: Nonviral Vectors for . by Mark A. Findeis Nonviral Vectors for Gene Therapy Methods and Protocols Humana Press Humana Press Edited by Mark A. Findeis Nonviral Vectors for Gene Therapy Methods and Protocols Synthesis. Backbone and Hyaluronic Acid Side Chains for DNA Carrier Atsushi Maruyama and Yoshiyuki Takei 1. Introduction Polycations have been used as nonviral gene carriers because the polycations and DNAs form. O., and Seymour, L. W. (1996) Characterization of vectors for gene therapy formed by self-assembly of DNA with synthetic block co-polymers. Hum. Gene Ther. 7, 2123–2133. 11. Kabanov, A. V. and