nonviral vectors for gene therapy, methods and protocols

1992 Use ofN-terminal modified polyL-lysine-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.. Recently, novel gene transfer techniques have bee

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

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 theassembling structure of DNA–copolymer complexes The dextran chains graftedonto PLL are found to reduce aggregation of the resulting complexes and toincrease the solubility of the complexes Furthermore, the grafting degree of thecopolymer affects the DNA conformation in the complex, allowing regulation ofDNA compaction The comb-type copolymers with a higher degree of graftinginduce little compaction of DNA and stabilize DNA duplexes and triplexes byshielding 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 specific carrier of DNA, if the copolymers are provided with cell-specific ligands.Hyaluronic acid (HA) is an unbranched high-molecular-weight polysaccharide

cell-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-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 inSECs, 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, weight HA was hydrolyzed, then the HAs were covalently coupled with ¡-aminogroups of PLL at their reducing end by reductive amination reaction

high-molecular-2 Materials

2.1 Enzymatic Hydrolysis of HA

1 High-molecular-weight HA (5.9 × 102kDa), obtained as its sodium salt (sodiumhyaluronate), 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 PeptideInstitute (Osaka, Japan)

2 Sodium borate buffer-NaCl: 0.1 M, pH 8.5, 0.4–1 M NaCl.

3 Sodium cyanoborohydride (NaBH3CN)

1 Chromatography pumping system operating at 1.0 mL/min

2 Size exclusion chromatography column(s)

3 NaNO3 (0.1 M) containing 5 mM sodium phosphate buffer, pH 8.0.

4 Na2SO4 (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 bySEC–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-µmfilter to remove the denatured enzyme The resulting HA fragments were obtained

by freeze-drying

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 isrecommended to estimate hydrolysis rate before a large-scale reaction The rate ofhydrolysis 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 isalso 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 aminationusing NaBH3CN as a reducing agent (Scheme 1) The reaction proceeded

through two steps First is the Schiff’s base (-CH=N-) formation between areductive (aldehyde) end of HA and primary amino groups of PLL Second isreduction of the Schiff’s base to form secondary amino groups (-CH2-NH).Although the Schiff’s base is unstable and reversible, its reduced product is anirreversible 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 precipitationwas observed, increase NaCl concentration PLL and HA may form aninterpolyelectrolyte 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 × 102kDa; 1 g) was hydrolyzed by hyaluronidase (20 mg) at 50°C

(mL)

4 Add NaBH3CN to the mixture and allow to stand at 40°C for 2 d Approximately

10 molar excess of NaBH3CN 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 isobtained by freeze-drying The resulting copolymer would be precipitated duringthe 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 peakarea of free HA, increase in peak of the copolymer and in molecular weight ofthe 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.

3.3 SEC–MALLS

SEC was carried out using a JASCO 880-PU pumping system (Tokyo, Japan) atthe 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) Asuitable 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 determinationusing the calibration curve based on molecular weight standard samples such aspolyethyleneglycol and pullulan is not reliable The choice of the mobile-phaserely on gel permeation chromatography (GPC) columns It is highly recommended

to provide a light-scattering (LS) detector system such as MALLS (Multianglelaser 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 NaNO3for HA fragment analysis and

0.2 M Na2SO4containing 5 mM sodium phosphate buffer (pH 8.0) for

copoly-mer analysis For typical analysis, 200 µL of each sample was picked up fromthe reaction mixture and injected into the columns Eluate was detected by arefractive index (RI) detector (830-RI, JASCO) and a MALLS detector RI and

LS signals were transferred to a computer to calculate number-average andweight-average molecular weight according to the instruction manual (WyattTechnology) for Dawn-DSP

3.4 1 H Nuclear Magnetic Research (NMR) Spectroscopic Analyses

Each copolymer was dissolved in D2O (Deuterium content: 99.95% Merck,

Darmstadt, Germany) containing 0.35 M NaCl.1H-NMR spectra (400 MHz) were

Fig 2 Time-course of coupling reaction between PLL and HA detected by SEC–MALLS

obtained by a Varian Unity 400plus spectrometer (Palo Alto, CA), at a probetemperature of 298 K The chemical shifts are expressed as parts/million usinginternal HDO molecules (b = 4.7 ppm in D2O) as a reference.

As shown in Fig 3, the 1H-NMR spectra of the comb-type copolymer showedthe characteristic signals of both PLL and HA moieties: PLL, b 1.4–1.8 (`, a,b-CH2), 3.0 (¡-CH2), 4.3 (_-CH); HA, b 2.0 (NAc-CH3), 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 type structure by combining enzymatic hydrolysis and the reductive amination

comb-Fig 3 1H NMR spectra of PLL (A), HA (B), and PLL-g-HA (C) in D2O For the

PLL-g-HA, D2O containing 0.35 M NaCl was used Reprinted with permission from

ref 19 Copyright 1998, American Chemical Society.

Table 1

In feed Copolymer CouplingPLL HA Mol wtb HA Contentc efficiencyd Yield

ChargeSample Mn/10 4 mg Mn/10 3 mg wt% Mn/10 4 Mw/Mn wt% Grafting-% ratio % %

a Reducing reagent, 0.3 M NaBH3CN; 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 (Mw/Mn) were determined by SEC–MALLS.

cDetermined 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.

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 Wagner, E., Cotten, M., Foisner, R., and Birnstiel, M L (1991) polycation-DNA complexes: the effect of polycations on the structure of the

Transferrin-complex and DNA delivery to cells Proc Natl Acad Sci USA 88, 4255–4259.

6 Huckett, B., Ariatti, M., and Hawtrey, A O (1990) Evidence for targeted genetransfer by receptor-mediated endocytosis: stable expression following insulin-

directed entry of neo into HepG2 cells Biochem Pharmacol 40, 253–263.

7 Trubetskoy, V S., Torchilin, V P., Kennel, S J., and Huang, L (1992) Use ofN-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted

gene delivery in mouse lung endothelial cells Bioconjugate Chem 3, 323–327.

8 Martinez-Fong, D, Mullersman, J E., Purchio, A F., Armendariz-Borunda, J.,and Martinez-Hernandez, A (1994) Nonenzymatic glycosylation of poly-L-lysine:

a new tool for targeted gene delivery Hepatology 20, 1602–1608.

9 Perales, J C., Grossmann, G A., Molas, M., Liu, G., Ferkol, T., Harpst, J., Oda,H., and Hanson, R W (1997) Biochemical and functional characterization ofDNA complexes capable of targeting genes to hepatocytes via the

asialoglycoprotein receptor J Biol Chem 272, 7398–7407.

10 Wolfert, M A., Schacht, E H., Toncheva, V., 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.

11 Kabanov, A V and Kabanov, V A (1995) DNA complexes with polycations for

the delivery of genetic material into cells Bioconjugate Chem 6, 7–20.

12 Maruyama, A., Katoh, M., Ishihara, T., and Akaike, T (1997) Comb-type

polycations effectively stabilize DNA triplex Bioconjugate Chem 8, 3–6.

13 Maruyama, A., Watanabe, H., Ferdous, A., Katoh, M., Ishihara, T., and Akaike, T.(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 copolymerand poly(D,L-lactic acid) Bioconjugate Chem 8, 735–742.

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

Ca2+-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 Takei, Y., Maruyama, A., Nogawa, M., Asayama, S., Ikejima, K., Hirose, M., et al.(1999) A novel gene delivery system for genetic manipulation of sinusoidal)endothelial cells by triplex DNA technology: evaluation of targetability and abil-

ity to stabilize triplex formation Hepatology 30, 298A.

19 Asayama, S., Nogawa, M., Takei, Y., Akaika, T., Maruyama, A (1998) Synthesis ofnovel polyampholyle comb-type copolymers consisting of poly (L-lysine) backbone

and hyaluronic acid side chains for a DNA carrier Bioconjugate Chem 9, 476–481.

From: Methods in Molecular Medicine, vol 65: Nonviral Vectors for Gene Therapy

Edited by: M A Findeis © Humana Press Inc., Totowa, NJ

particu-so on, which electrostatically form a complex with the negatively chargedDNA, which can be taken up by the cells Furthermore, targeted gene transferhas also been realized by modification of the gene carriers using cell-targetingligands such as asialoorosomucoid, transferrin, insulin, or galactose

Recently, novel gene transfer techniques have been reported, in which anamphiphilic _-helical peptide, containing cationic amino acids is used as a

gene carrier into cells Wyman et al (1) employed a peptide, KALA

(WEAK-LAKA-LAKA-LAKH-LAKA-LAKA-LKAC-EA), which is derived from thesequence of the N-terminal segment of the HA-2 subunit of the influenza virushemagglutinin involved in the fusion of the viral envelope with the endosomalmembrane This peptide showed several functions in the transfection process,e.g., condensing DNA and causing an endosome-membrane perturbation,which enables it to deliver the incorporated DNA to the cytosol, which isessential for efficient transfection Similarly, the authors also found thetransfection technique, which is mediated by some amphiphilic _-helicalpeptides (e.g Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NHCH3[46] and

KLLK-LLLK-LWKK-LLKL-LK [Hel]) as shown in Table 1 (2–5) After that,

for the purpose of refining of the peptide structure, we investigated theinfluence of the peptide chain length on gene transfer ability As a result, 16and 17 amino acid residues were sufficient to form aggregates with the DNA,

and transfer the DNA into the cells in the deletion series of 46and Hel, respectively

(Table 1; 6) In addition, the authors succeeded in constructing a multiantennary

galactose-modified peptide containing four galactose residues that serve for

efficient binding to the asialoglycoprotein receptor on hepatoma cells (7).

This chapter focuses on synthesis of the peptides and a method of gene fer using them As is well known, a peptide is readily synthesized because ofthe development of an automatic peptide synthesis apparatus and reagents forsynthesis From this point of view, it is expected that the gene transfer methodmediated by the peptide is easily accepted by many researchers taking part inthe gene therapy study

trans-2 Materials

2.1 Peptide Synthesis

1 Peptide synthesis apparatus (ABI 431A, PE Biosystems)

2 Fmoc-Lys(Boc) preloaded Wang resin, Rink amide resin (100–200 mesh)(Calbiochem-Novabiochem, CA)

3 Fmoc protected amino acids,

2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetrame-thyluronium hexafluorophosphate (HBTU), N,N,-diisopropylethylamine,

NMP, dichloromethane (DCM), piperidine (Watanabe Chemical, Hiroshima, Japan)

4 Thioanisole, m-Cresol, ethandithiol, trifluoracetic acid (TFA), acetonitrile (Wako

Chemicals, Osaka, Japan)

5 High-performance liquid chromatography (HPLC) apparatus (Hitachi L7100System, Tokyo, Japan)

6 Reverse-phase (RP)-HPLC column (YMC-Pack C4, q10× 150 mm, Kyoto, Japan)

7 Matrix-assisted Laser Desorption Ionization-Time of Flight-Mass Spectra(MALDI TOF-MS) apparatus (Voyager DE STR, PE Biosystems)

2.2 Preparation of Plasmid DNA

1 Plasmid DNA, which contains a luciferase gene and SV40 promoter (PicaGenecontrol vector, PGV-C), was purchased from Toyo Ink (Tokyo, Japan)

2 Plasmid DNA (pCMVluc), containing a luciferase gene under control of

cytomegalovirus enhancer/promoter, was prepared by removing the BglII and

Table 1

Structures of Amphiphilic _-Helical Peptides

Chain CationicPeptide Sequence length charge

46 Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH2 24 6

4668 Ac-LARL-LRAL-LRAL-LRAL-NH2 16 4Hel KLLK-LLLK-LWKK-LLKL-LK 18 7Hel61 LLK-LLLK-LWKK-LLKL-LK 17 6

HindIII insert of the plasmid PGV-C (Toyo Ink), and ligating with the BglII

and HindIII fragment from the pRc/CMV (Invitrogen), which contains

cytomegalovirus promoter

3 Closed circular plasmid DNA was purified by ultracentrifugation in cesium ride gradients The plasmid preparations showed a major band of closed circularDNA and minor amount (<20%) of nicked plasmid

chlo-2.3 Cell Culture and Gene Transfer into Cells

1 COS-7 cells (a monkey kidney cell line, RCB accession no RCB0539), HeLa cells(a human cervix, RCB accession no RCB0271) and CHO cells (a Chinese hamsterovary, RCB accession no RCB0285) (RIKEN Cell Bank, Tsukuba, Japan)

2 HuH-7 cells (a human hepatoma cell line, JCRB accession no JCRB0403)(Health Science Research Resources Bank, Osaka, Japan)

3 Dulbecco’s modified Eagle’s medium, RPMI 1640, media supplements, and inactivated fetal calf serum (IWAKI Glass, Chiba, Japan)

heat-4 PicaGene luminescence kit (Toyo Ink)

5 Luminometer (Maltibiolumat LB9505, Berthold, Germany)

3.1.1 Elongation of Peptide Chain on Resin

Peptides can be manually synthesized by the stepwise elongation of Fmocprotected amino acid on Rink amide resin (for 46; 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin, 0.43 mmol/g resin) or Fmoc-Lys(Boc)

preloaded Wang resin (for Hel; Fmoc-Lys(Boc)-p-benzoyloxy alcohol resin,

0.58 mmol/g resin) in 0.1 mmol scale as described by Fields et al (8), and in

Catalog & Peptide Synthesis Handbook of Calbiochem-Novabiochem TheFmoc amino acid derivatives used are as follows: Ala, Arg(Pbf), Leu,Lys(Boc), and Trp The coupling protocol is shown as follows:

6 Coupling of amino acid; Fmoc-amino acid (0.3 mmol), HBTU (0.3 mmol), HOBt

(0.3 mmol), and N,N,-diisopropylethylamine (DIEA) (0.6 mmol) in 3 mL

NMP:DMF (1:1) (15 min)

7 Wash: 2 mL DMF (3×)

8 Kaiser test (9): When the coupling is incomplete, the protocol is repeated from step 6.

As a matter of course, it is possible to elongate the peptide chain using automaticpeptide synthesizer (PE Biosystems, ABI 431A) In this case, the peptides are alsosynthesized by FastMoc method in 0.1 mmol scale Even if single coupling of aminoare applied for all elongation steps, satisfactory peptides in purity are obtained In thecase of modifying by acetyl group to N-terminal of peptide, 46, the Fmoc-deprotectedpeptide on resin is treated with 1 mmol acetic anhydride in 2 mL NMP for 20 min.

After the resin is washed by DCM and methanol, they are dried in vacuo.

3.1.2 Deprotection and Cleavage of Peptide Resin

The protecting groups and the resin are removed with TFA (2.0 mL) in the

presence of m-cresol (60 µL), ethandithiol (180 µL), and thioanisole (360 µL),

in a round flask After swirling at room temperature for 60 min, the resin isremoved by filtration under reduced pressure and washed by TFA Twentymilliliters of cold diethylether is added to the filtrate, then, the flask can becooled with ice to further assist precipitation Crude peptide is isolated by fil-tration under reduced pressure and washed by cold diethylether

The crude peptides can be purified by RP-HPLC on a YMC-Pack C4 column(q10× 250 mm) with a linear gradient of water–acetonitrile containing 0.1% TFA.Peptide 4668 and Hel61 are eluted at about 80 and 55% of acetonitrile, respec-tively The authors recommend that if possible, the crude peptide be passed through

a column of Sephadex G-10 or G-15 (q10× 250 mm) with 10% acetic acid beforepurification by RP-HPLC to avoid damaging the HPLC column The peptide frac-tions are collected, then lyophilized The yields obtained after purification are about

80 (34 µmol) and 100 mg (36 µmol) in the cases of 4668 (as 4TFA salt) and Hel61(as 6TFA salt), respectively The purified peptides can easily be identified byMALDI-TOF-MS ([M + H]+ = 1874.4 [4668], 2105.7 [Hel61])

3.2 Preparation of Peptide–DNA Complex

Complex of peptide and DNA is prepared by mixing 2.5 µg plasmid DNA(PGV-C) with the peptides at peptide:DNA charge ratio of 2.0 in serum-freemedium The authors protocol is shown as follows:

1 Prepare the DNA solution: 2.5 µg plasmid DNA in 250 µL serum-free culturemedium

2 Prepare the peptide solution: 1.6 mM (as cationic groups) peptide aqueous

solu-tion Practically, 930 µg 4668 • 4TFA or 743 µg Hel61 • 6TFA are dissolved in1.0 mL sterilized water

3 10 µL Peptide solution is added to 250 µL plasmid DNA solution

4 Stand for 15 min at room temperature

3.3 Gene Transfer Protocol into Cultured Cells

1 Plate cells in 24-well tissue culture dishes (q16 mm) at 1 × 105cells/well andgrow overnight in an atmosphere of 5% CO at 37°C

2 Wash twice with 1 mL serum free medium.

3 The peptide–DNA complex as described above is poured gently to the cells

4 After incubation for 3 h at 37°C, 1 mL medium containing serum is added

5 After incubation for 12 h at 37°C, the medium is replaced with 1 mL freshmedium 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 assayusing bovine serum albumin as a standard The light unit values shown in thefigures represent the specific luciferase activity (relative light units/mg protein),which is standardized for total protein content of the cell lysate The measure-ment 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 acommercially available gene transfer agent, we employed Lipofectin (Gibco-BRL)

As a result, the efficiencies of the peptides were similar to or about 10-fold lowerthan those of Lipofectin Except for the case of needing a large amount of expres-sion product, it can be said that the peptides are enough to use as a gene carrier Onthe other hand, the peptides showed different efficiencies depending on the celllines These peptides can be chosen according to the cell lines

The authors also evaluated cytotoxic activities of the peptide–DNAcomplexes, using Alamer BlueTM, under the same conditions as those in thetransfection procedure As a result, little cytotoxic activity of the complexescould be observed However, when the complex is prepared at peptide:DNAcharge ratio of 4 and more, considerable cytotoxic activities are observed,which the authors consider to result from membrane perturbation activities

originated from the amphiphilic structures of the peptides (3,5).

3.4 Active Targeting of Gene into Hepatoma Cells Using Galactose Modification of Peptide

As described in the Introduction, peptide is easily available because of thedevelopment of its synthetic technology Therefore, this allows design andsynthesis of functional gene carrier molecules such as carbohydrate-modifiedpeptide for targeted gene delivery Furthermore, peptide-based gene carrierenables construction of well-defined molecules, which cannot be achieved bypolymer-based molecule Here is introduced a synthesis of a multiantennarygalactose-modified peptide and its application to a human hepatoma cell line.3.4.1 Preparation of Multiantennary Galactose-Modified Peptide

The synthesis method is described as follows and is summarized in Fig 2.

1 _-Helical peptide portion can be synthesized by ordinary Fmoc solid-phase

method on Rink amide resin (see Subheading 3.1.1.).

2 After coupling Fmoc–2-(2-aminoethoxy) ethanol as a linker, Lys(Fmoc) is coupled twice Fmoc groups of secondary coupled Fmoc-`Ala-Lys(Fmoc) are removed by piperidine At this step, the peptide has four aminogroups in the molecule

Fmoc-`Ala-3 After cleavage from resin and deprotection, the peptide is purified by RP-HPLC

as similar condition to the case of peptide 46 (see Subheading 3.1.2.)

4 The amino groups of the peptide can be modified with lactose by aldimineformation, followed by reduction with NaBH3CN of the secondary amines asfollows The purified peptide (11 µmol, 50 mg as 10TFA salt) is mixed with asolution of lactose (530 µmol, 190 mg as monohydrate) in 500 µL 10 mM aqueous

Fig 1 Gene transfer efficiencies of the peptides into several cell lines

sodium acetate, pH 5.0, at 37°C, then NaBH3CN (44 µmol, 2.8 mg each) is added

to the solution at 12-h intervals After total incubation for 60 h at 37°C, the ant galactose-modified-peptide is purified by RP-HPLC Yield is 20 mg (38%)

result-5 Identification is performed by MALDI-TOF-MS ([M + H]+ = 4816.7) Thepeptide concentration in solution is determined from UV-absorbance of Trp at

280 nm in a buffer containing 6 M Gu • HCl (¡ = 5500).

3.4.2 Gene Transfer Using Galactose-Modified Peptide into

Hepatoma Cell Line, HuH-7

Protocol for gene transfer into HuH-7 cells, a human hepatoma cell line, is

similar to that described in Subheadings 3.2 and 3.3 Complex of peptide and

DNA is prepared by mixing 2.5 µg plasmid DNA (pCMVluc) with Gal4–46 atpeptide:DNA charge ratio of 2.0 in serum free medium

1 Prepare the DNA solution: 2.5 µg plasmid DNA in 250 µL serum free RPMI 1640.Fig 2 Outline of synthesis of galactose-modified peptide, Gal4–46

2 Prepare the peptide solution: 1.6 mM (as cationic groups) peptide aqueous

solu-tion Practically, 1.5 mg Gal4–46• 10TFA is dissolved in 1.0 mL sterilized water

3 10 µL peptide solution is added to 250 µL plasmid DNA solution

4 Stand for 15 min at room temperature

5 The peptide–DNA complex as described above is poured gently on to the cells,which are washed twice with 1 mL serum-free RPMI 1640, beforehand Follow-

ing procedures are same to steps 4–7 in Subheading 3.3.

As a result of measurement of luciferase activity in HuH-7 cells, the activity

of Gal4–46was 300-fold higher than that of 46 In addition, the authors couldconfirm that the DNA complex of Gal4–46was internalized into the cells via

the asialoglycoprotein receptor (see Subheading 3.5.).

This chapter introduced a galactose-modified peptide containing aminoethoxy)ethanol and `Ala as a linker, which allows the galactose residues to

2-(2-be easily recognized by the receptor However, the authors found that a Gal4–46derivative without any linker showed a high efficiency into HuH-7 cells, as similar

to that of Gal4–46 Insertion of linker into the peptide system is not always sary for recognition of the galactose residues by the receptor

neces-3.5 Commentary

In the authors’ series of studies, it has become clear that the hydrophobic region

on the amphiphilic structure of the peptides plays an important role in binding tothe plasmid DNA and formation of aggregates with the DNA It is likely that thehydrophobic region of the peptides induces stable oligomers with a well-definednumber of monomers by self-association with their hydrophobic faces in aqueoussolution As a result, the oligomer would behave like a polycation, which can form

aggregates with DNA (Fig 3) Furthermore, the authors indicate that the

hydro-phobic region is also important for the disruption of the endosomal membrane inthe cell, which can transfer the incorporated DNA to cytosol and prevent the degra-

dation of the DNA in the lysosomal vesicles (3,5).

In order to clear detail translocation pathway of the DNA in the cell, theauthors evaluated effects of several endocytosis inhibitors on the gene transferefficiencies of peptide 46 Treatment with cytochalasin B, which depolymerizesthe microfilaments of actin and blocks the uncoated pit-mediated endocytosis

(macropinocytotic process) (10), reduced to 25% of the original efficiency of

46; no effect was observed in the case of treatment with chlorpromazine, an

inhibitor of clathrin-dependent, receptor-mediated endocytosis (11)

Further-more, N-ethylmaleimide (NEM), which inhibits fusion between endosomes at

an early stage of the endocytic pathway (12), reduced to 50% of the original

efficiency, and the microtubule-depolymerizing agent, nocodazole, whichinterferes with transport from the early to late endosome mediated by endocytic

carrier vesicles (13), increased the efficiency of the peptide by sixfold From

these results, it is reasonable to suppose that the complexes of peptide 46andplasmid DNA were internalized by macropinocytotic process, and a part of thecomplexes could be translocated from the endosomal compartment to the cytosol

during an early endosome step (Fig 3) However, the other part of the complexes

would be translocated into degradation compartments such as late endosome andlysosome, where the complex could no longer be translocated to the cytosol Inorder to enhance gene transfer efficiency, it will be necessary to consider activetranslocation to the cytosol of the DNA complex Because there is now no infor-mation for translocation into nucleus of the complex, elucidation of this transfermechanism in the cell will give a clue to construct a novel gene carrier withhigher efficiency

The authors have also studied the transfer pathway of the DNA complexwith Gal4–46 At first, the competitive effects of asialofetuin, which is internal-ized into hepatoma cells via the asialoglycoprotein receptor-mediated endocy-tosis, and fetuin, which is not recognized by the receptor, on the transferefficiencies of the peptides were examined As a result, the transfer efficiency

of Gal4–46is reduced to 1% of the original efficiency in the presence of theasialofetuin, but no effect was observed in 46 However, fetuin showed weakeffect compared with asialofetuin

In addition, the authors evaluated effects of several endocytosis inhibitors

on the gene transfer efficiencies of Gal4-46 Treatment with chlorpromazinesignificantly reduced the efficiency; no significant reduction was observed inthe case of treatment with cytochalasin B This result suggested that the

Fig 3 Formation of peptide–DNA complex and its transfer pathway into cell

internalization of Gal4–46was mediated by the clathrin-dependent, mediated endocytosis Furthermore, NEM reduced to 50% of the originalefficiency, and nocodazole increased the efficiency by twofold From theseresults, DNA complex with the galactose modified peptide, Gal4–46, would betranslocated from the endosomal compartment to the cytosol during an earlyendosome step as in the case of 46.

receptor-Finally, to apply these galactose-modified peptides for targeted genedelivery in vivo, it is necessary to further examine the cell selectivity usingseveral cell lines, stability in blood, capture by the reticuloendothelial system,and so on When these points are solved, this delivery system will be one of thepowerful tools for gene therapy

References

1 Wyman, T B., Nicol, F., Zelphati, O., Scaria, P V., Plank, C., and Szoka, F C.(1997) Design, synthesis, and characterization of a cationic peptide that binds to

nucleic acids and permeabilizes bilayers Biochemistry 36, 3008–3017.

2 Ohmori, N., Niidome, T., Hatakeyama, T., Mihara, H., and Aoyagi, H (1998)Interaction of _-helical peptides with phospholipid membrane: effects of chain

length and hydrophobicity of peptides J Peptide Res 51, 103–109.

3 Niidome, T., Ohmori, N., Ichinose, A., Wada, A., Mihara, H., Hirayama, T., andAoyagi, H (1997) Binding of cationic _-helical peptides to plasmid DNA and

their gene transfer abilities into cells J Biol Chem 272, 15,307–15,312.

4 Kiyota, T., Lee, S., and Sugihara, G (1996) Design and synthesis of amphiphilic_-helical model peptides with systematically varied hydrophobic-hydrophilic

balance and their interaction with lipid- and bio-membranes Biochemistry 35,

13,196–13,204

5 Ohmori, N., Niidome, T., Kiyota, T., Lee, S., Sugihara, G., Wada, A., Hirayama,T., and Aoyagi, H (1998) Importance of hydrophobic region in amphiphilic struc-tures of _-helical peptides for their gene transfer-ability into cells Biochem

Biophys Res Commun 245, 259–265.

6 Niidome, T., Takaji, K., Urakawa, M., Ohmori, N., Wada, A., Hirayama, T., andAoyagi, H (1999) Chain length of cationic _-helical peptide sufficient for gene

delivery into cells Bioconjugate Chem 10, 773–780.

7 Niidome, T., Urakawa, M., Sato, H., Takahara, Y., Anai, T., Hatakayama, et al.(2000) Gene transfer into hepatoma cells mediated by galactose-modified alpha-

helical peptides Biomaterials 21, 1811–1819.

8 Fields G B and Noble R L (1990) Solid phase peptide synthesis utilizing

9-fluorenylmethoxycarbonyl amino acids Int J Peptide Protein Res 35, 161–214.

9 Kaiser, E., Colescott, R L and Bossinger, C D (1970) Color test for detection of

free terminal amino groups in the solid-phase synthesis of peptides Anal.

Biochem 34, 595–598.

10 Paccaud J.-P., Siddle K., and Carpentier J.-L (1992) Internalization of the human

insulin receptor The insulin-independent pathway J Biol Chem 267, 13,101–13,106.

11 Orlandi P A and Fishman P H (1998) Filipin-dependent inhibition of choleratoxin: evidence for toxin internalization and activation through caveolae-like

domains J Cell Biol 141, 905–915.

12 Rothman J E (1994) Mechanisms of intracellular protein transport Nature

372, 55–63.

13 Lemichez E., Bomsel M., Devilliers G., vanderSpek J., Murphy J R., Lukianov

E V., Olsnes S., and Boquet P (1997) Membrane translocation of diphtheria toxin

fragment A exploits early to late endosome trafficking machinery Mol Microbiol.

23, 445–457.

From: Methods in Molecular Medicine, vol 65: Nonviral Vectors for Gene Therapy

Edited by: M A Findeis © Humana Press Inc., Totowa, NJ

3

Supramolecular Self-Assembly of Poly(ethylene

with Plasmid DNA

Joon Sig Choi and Jong Sang Park

1 Introduction

Research and development related to nonviral gene carriers comprisingchemically synthesized molecules has increased enormously during the pastdecade Polycationic polymers and cationic lipids have constituted the mainthemes of the studies Various polymers from synthetic to naturally occurringones have been introduced and tested for their suitability in the field of genetherapy Several cationic polymers were found to be promising but their intrin-sic drawbacks, such as solubility, 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 havebeen tested for their potential utility and have exhibited high transfection

efficiency in vitro and in vivo (2,3) However, these dendrimers have not yet

overcome the problems of solubility of the complex with DNA and cytotoxicity.Block copolymers containing poly(ethylene glycol) (PEG) have been used formany drug carriers because of their high solubility in water, nonimmunogenic-

ity, and improved biocompatiblity (4) PEG has been coupled to polycationic

polymers (e.g., poly-L-lysine [PLL], 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 blockcopolymer which is capable of polyionic complex formation with plasmid DNAvia supramolecular self-assembly Linear PEG was coupled to the globular

macromolecule, poly(L-lysine) dendrimer by the repetitive liquid-phase peptide

synthesis method (10) Poly(L-lysine) dendrimer (11–13) is another

polycationic dendrimer containing a large number of surface amines and isconsidered to be capable of electrostatic interaction with polyanions, such as

nucleic acids (14) The enhanced aqueous solubility of the complexes is an

advantage compared to that of homopolymer polycations and cationic lipids.This type of self-assembly is interesting, from both theoretical and practicalpoints of view in designing polymers for gene delivery vectors, because it canserve as a suitable model for polyionic complex formation of other hybrid blockcopolymers with DNA

2 Materials

2.1 Synthesis

1 Methoxypoly(ethylene glycol) amine, mol wt 3400 (Shearwater Polymer)

2 N-hydroxybenzotriazole (HOBt).

3 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)

4 N-_-N-¡-di-Fmoc-L-lysine (AnaSpec, San Jose, CA)

2 Ultra-pure water (Milli-Q Biocel system)

2.3 Agarose Gel Electrophoresis

1 5X HEPES-buffered saline (HBS): 0.1 M HEPES, 0.75 M NaCl, pH 7.4.

2 6X Loading buffer: 0.25% bromophenol blue, 0.25% xylene cyanol FF, 15%Ficoll (Type 400; Pharmacia) in water

3 5X TBE buffer: Tris 27 g, boric acid 14 g, 0.5 M EDTA, pH 8.0, 10 mL in 500 mL

of water

4 0.7% Agarose in TBE buffer containing 0.5 µg/mL ethidium bromide

2.4 Atomic Force Microscopy

1 Freshly split mica

2 Ultra-pure water

3 Whatman 3MM paper

2.5 DNase I Protection Assay

1 DNase I: 8.9 U/µL, 50% glycerol, 20 mM sodium acetate buffer, pH 6.5, 5 mM

CaCl2, 0.1 mM phenylmethylsulfonyl fluoride.

2 Stop solution: 4 M ammonium acetate, 20 mM EDTA, 2 mg/mL glycogen.

3 1% Sodium dodecyl sulfate

4 Tris-EDTA saturated phenol

10 0.7% Agarose in TBE buffer containing 0.5 µg/mL ethidium bromide

2.6 Water Solubility Test

2 Sterilized Dulbecco’s minimum essential medium

3 10% Fetal bovine serum

3.1 Synthesis of Hybrid Block Copolymer

1 The overall synthesis scheme is outlined in Fig 1 Prepare the reaction mixture

in anhydrous DMF: mPEG-amine 0.15 g (30 µmol) (see Note 1) Synthesis scale

for each step is described in Table 1.

2 After the coupling reaction reaches completion, precipitate the mixture with a10-fold excess of cold ether and further wash 2× with ether (see Note 2).

3 Deprotect the Fmoc group of lysine using 30% piperidine

4 Precipitate in cold ether and wash 2× with excess ether (see Note 3).

5 Dry the precipitates in vacuo and prepare for further coupling reaction.

6 Repeat the coupling and deprotection reactions 4× (see Note 4).

7 Solubilize the fourth generation copolymer in water

8 Dialyze for 1 d against water using Spectra/Por dialysis membrane (mol wt cut-off6–8 kDa) and filter the solution with 0.45-µM syringe filter.

9 Collect the final product by freeze-drying

Fig 1 Outline of stepwise liquid phase synthesis of mPEG-b-PLLD.

3.2 MALDI-TOF-MS

1 Prepare the stock solution in water: polymer solution, 2–4 mg/mL; DHB solution

10 mg/mL

2 Mix the two solutions: polymer solution: DHB matrix solution = 1: 9 µL or 2: 8 µL

3 Pipet about 1 µL mixture and load onto the plate (see Note 5).

4 Dry in vacuo (see Note 6).

5 Characterize the linear polymer/dendrimer block copolymer by MALDI-TOF-MS

(see Fig 2).

3.3 Agarose Gel Electrophoresis

1 Mix the polymer and pSV-`-gal plasmid DNA in 15 mM HEPES buffer, 0.15 MNaCl, pH 7.4 Incubate at room temperature for 30 min

2 Prepare 0.7% agarose gel containing ethidium bromide (0.5 µg/mL gel)

3 After 30 min, mix with 6X 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 (see Note 8).

Charge ratio (z) 1 2 4

Polymer solution (µg/µL) 0.03 0.06 0.12

5µL 5 µL 5 µLDNA solution

3.5 DNase I Protection Assay

1 Add the copolymer to 4.0 µg of plasmid DNA at various charge ratios from 0 to

4, in 50 µL of 20 mM HEPES buffer, 0.15 M NaCl, pH 7.4

2 Incubate for 30 min at room temperature

Fig 2 MALDI-TOF-MS spectrum of the copolymer The spectrum was obtained

on a PerSeptive Biosystems instrument in the linear mode at 20 kV The Mwand Mnvalues of this copolymer were 7594 and 7553, respectively (Mw/Mn = 1.01)

Fig 3 Analysis of complex formation at various charge ratios by agarose gel

electro-phoresis (A) mPEG-PLLD (generation 3) 1.0 µg pSV-`-gal plasmid DNA only (lane 1),

charge ratio of copolymer: DNA = 0.5, 1, 2, and 4 (lanes 2, 3, 4, and 5, respectively) (B)

mPEG-PLLD (generation 4) 1.0 µg pSV-`-gal plasmid DNA only (lane 1), charge ratio of copolymer: DNA = 1, 2, 3, 5, and 6 (lanes 2, 3, 4, 5, and 6, respectively).

Fig 4 The atomic force microscopy images of the mPEG-block-PLLD/pSV-`-gal

complex Charge ratios of copolymer: DNA = 1, 2, and 4 (A, B, and C) The image

mode was set to tapping mode The white color indicates a height more than the

desig-nated nm above the mica surface The x and y dimensions are scaled as shown.

3 Further incubate the mixtures in the presence of 8.9 U of DNase I for 20 min atroom temperature.

4 To each mixture, add 75 µL stop solution and keep each tube on ice

5 After addition of 37 µL 1% sodium dodecyl sulfate, extract DNA with Tris-EDTAbuffer-saturated phenol/chloroform

6 Precipitate the DNA pellet by adding pure ethanol

7 Dissolve the precipitated DNA in Tris-EDTA buffer and subject to 0.7% agarose

gel electrophoresis (see Fig 5).

3.6 Solubility Test of the Polyplex

1 Add the polymer to 26 µg plasmid DNA at a charge ratio of 4 in 0.5 mL 20 mM

HEPES buffer, 0.15 M NaCl, pH 7.4 (see Note 11).

2 Incubate for 30 min at room temperature

3 Centrifuge each tube for 5 min at 13,000 rpm (16,816g), 10°C

4 Measure the absorbance of the supernatant at 260 nm

5 Calculate the absorbance percentage compared to that of DNA only solution (see

Fig 6).

3.7 In Vitro Cytotoxicity Assay

1 Seed the 293 cells (human embryonic kidney cell lines) in 96-well microplates at

a density of 10,000 cells/well in 0.1 mL growth medium DMEM containing 10%fetal bovine serum

2 Introduce the polymer to each well and incubate for 48 h

3 Remove the old medium and replace with new growth medium containing MTT(26µL 2 mg/mL stock solution/each well) (see Note 12).

4 Incubate the plate for an additional 4 h at 37°C

5 Remove each medium prior to the addition of 150 µL of dimethylsulfoxide

6 Mix up and down using eight-channel pipet

7 Measure the absorbance at 570 nm using a microplate reader (Molecular Devices,Menlo Park, CA)

8 Calculate the absorbance percentage relative to that of untreated control cells

(see Fig 7).

4 Notes

1 It is important to get a MALDI signal for the starting material, mPEG-amine

(weight average molecular weight [Mw]= 5757, number average molecular weight

[Mn]= 5697, Mw/Mn= 1.01, determined by MALDI-TOF mass spectrum).Usually, the specified molecular weight of commercial PEG differs from theexperimental value

2 The progress of each reaction was monitored by ninhydrin test and 1H-nuclearmagnetic resonance until completed 1H-nuclear magnetic resonance (D2O)b 1.64(broad multiplet [brm], [CH2]3), 3.08 (brm, CH2-N), 3.39 (siglet [s], CH3-O),3.68 (s, CHCH-O), 4.25 (brm, COCH-N)

3 In order to remove small traces of excess reagents, the precipitate wasrecrystalized in pure ethanol Subsequently, the product is obtained as a pure-white crystalline powder.

4 Sometimes, insoluble precipitates were formed because of low solubility It isrecommended to conduct the fourth coupling reaction with half of the totalamount of synthesized third generation copolymer

5 For polymers, about 100 pmol is required to get an effective MALDI signal

Fig 5 Stability of mPEG-block-PLL–plasmid DNA complexes to DNase I at various charge ratios Undigested plasmid DNA (lane 1), Charge ratio of copolymer: DNA = 0, 0.5, 1, 2, and 4 (lanes 2, 3, 4, 5, and 6, respectively) The positions of the open circular

(oc), linear (li), and supercoiled (su) forms are indicated on the right

Fig 6 Water-solubility test of some polyplexes The complex of mPEG-PLLDwith plasmid DNA showed much more increased solubility than that of PLL orpolyethylenimine

6 Usually, it is better to dry the mixture in vacuo, rather than just air-drying for the

preparation of an adequate specimen for MALDI-TOF-MS

7 In the case of mPEG-PLLD (G = 3), any complete retardation that results fromfull complexation between polymer and DNA was not observed even though thecharge ratio of polymer:DNA was increased to 4

8 1.89 × 1015negative charges are present per 1.0 µg plasmid DNA; the copolymerhas 1.26 × 1015 charges/1.0 µg

9 Usually, about 10 ng plasmid DNA is required for the AFM imaging The amount

of the DNA seems to be critical for obtaining the best image

10 Try to load the complex solutions in the center of mica surface After 1–2 min, donot disturb the center and remove the residual solution carefully

11 19.2 kDa PLL and 25 kDa polyethylenimine were used as control reagents Allthe polymers were mixed with DNA at a charge ratio of 4

12 MTT solution is sensitive to light After filtering the stock solution through a 0.2 µmsyringe filter, it must be preserved in a light-protected tube It is advisable to wrap thetube with aluminum foil

References

1 Ledley, F D (1995) Nonviral gene therapy: the promise of genes as

pharmaceu-tical products Hum Gene Ther 6, 1129–1144.

2 Kukowska-Latallo, J F., Bielinska, A U., Johnson, J., Spindler, R., Tomalia, D A.,and Baker, J R., Jr (1996) Efficient transfer of genetic material into mammalian

cells using Starbust polyamidoamine dendrimers Proc Natl Acad Sci USA 93,

4897–4902

Fig 7 Effect of PLL and mPEG-PLLD (G = 4) on 293 cells viability Relativeviability is expressed considering the absorbance at 570 nm intact cells as 100% Eachdata point is the average ± SD of six different experiments

3 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 USA 92,

7297–7301

4 Kataoka, K., Kwon, G S., Yokoyama, M., Okano, T., and Sakurai, Y (1993)

Block copolymer micelles as vehicles for drug delivery J Controlled Release 24,

6 Kataoka, K., Togawa, H., Harada, A., Yasugi, K., Matsumoto, T., and Katayose,

S (1996) Spontaneous formation of polyion complex micelles with narrow bution from antisense oligonucleotide and cationic block copolymer in physiologi-

distri-cal saline Macromolecules 29, 8556–8557.

7 Kabanov, A V., Vinogradov, S V., Suzdaltseva, Y G., and Alakhov, V Y (1995)Water-Soluble block polycations as carriers for oligonucleotide delivery

Bioconjugate Chem 6, 639–643.

8 Nguyen, H -K., Lemieux, P., Vinogradov, S V., Gebhart, C L., Guérin, N.,Paradis, G., et al (2000) Evaluation of polyether-polyethyleneimine graft copoly-

mers as gene transfer agents Gene Ther 7, 126–138.

9 Lee, R J and Huang, L (1996) Folate-targeted, anionic liposome-entrapped

polylysine-condensed DNA for tumor cell-specific gene transfer J Biol Chem.

271, 8481–8487.

10 Bayer, E and Mutter, M (1979) The Liquid-phase method for peptide synthesis,

in The Peptides (Gross, E and Meienhofer, J., eds.), vol 2, Academic, New York,

J Chem Soc Chem Commun 1869–1872.

13 Chapman, T M., Hillyer, G L., Mahan, E J., and Shaffer, K A (1994)

Hydraamphiphiles: novel linear dendritic block copolymer surfactants J Am.

Chem Soc 116, 11,195–11,196.

14 Choi, J S., Lee, E J., Choi, Y H., Jeong, Y J., and Park, J S (1999)Poly(ethylene glycol)-block-poly(L-lysine) dendrimer: novel linear polymer/dendrimer block copolymer forming a spherical water-soluble polyionic complex

with DNA Bioconjugate Chem 10, 62–65.

From: Methods in Molecular Medicine, vol 65: Nonviral Vectors for Gene Therapy

Edited by: M A Findeis © Humana Press Inc., Totowa, NJ

membranes, a carrier system is required for transfection (1–4) Cationic

poly-mers, which condense DNA by ionic interaction, form a promising class ofnonviral transfection agents Well-known examples of these polymers are DEAEdextran, poly(L-lysine), poly(ethylenimine), and poly(2-[dimethylamino]ethyl

methacrylate) (pDMAEMA) (5–10) In order to achieve transfection, a plasmid

must be delivered into the nucleus, which requires cellular uptake of polymer–

DNA complexes, generally referred to as “polyplexes” (11), which most likely

occurs via endocytosis, followed by endosomal escape and transport to thenucleus The polyplex must dissociate, either in the cytosol or in the nucleus,which may be a critical step in the transfection process

pDMAEMA (Fig 1), a water-soluble cationic polymer, may form

self-assembled stable nanoparticles with plasmids at a polymer:plasmid ratio above

2 (w/w) (8–10) The positively charged polyplexes have a size of ~150–200 nm

(8–10) The transfection efficiency vs the pDMAEMA:plasmid ratio forms a

bell-shaped curve, with a maximum at 3–6 (w/w), depending on the transfectedcell type At low pDMAEMA:plasmid ratios, addition of more polymer to thecomplexes results in smaller complexes with higher transfection efficiency; athigher pDMAEMA:plasmid ratios, the slight cytotoxicity of the polymer

probably results in cell death (8–10) A pronounced effect of the molecular

weight of the polymer on the transfection efficiency is observed An increasing

molecular weight results in an increasing number of transfected cells (9) Dynamic

light scattering experiments show that high-molecular weight polymers (mol wt

> 300 kDa) are able to condense DNA effectively (particle size, 150–200 nm) Incontrast, when plasmid is incubated with low-molecular-weight pDMAEMA, largecomplexes are formed (size 0.5–1.0 µm) Using optimal conditions, the degree oftransfection in vitro (approx 30% of the treated cells are transfected) is higher thanthat achieved with commercially available cationic lipids such as 1,2-dioleoyl-

3-trimethylammonium-propane (DOTAP) and Lipofectamine (12) Cells grown

in vivo can be transfected ex vivo with pDMAEMA-based polyplexes with an

overall transfection efficiency of ~1–2% (13).

To further improve transfection performance, random copolymers weresynthesized Random copolymers of DMAEMA with ethoxytriethylene glycolmethacrylate (triEGMA), N-vinylpyrrolidone (NVP), methyl methacrylate(MMA), and methacrylic acid of different molecular weights and composi-tions (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 of DMAEMA with triEGMA, NVP, and MMA, thepolymer:plasmid ratio at which small complexes (size 200 nm) are formed

increases with the mol fraction of the comonomer (14,15) A copolymer with

20 mol% MMA shows a reduced transfection efficiency and a substantialincreased cytotoxicity compared with a homopolymer of the same molecular

weight (14) On the other hand, for triEGMA, NVP, and methacrylic acid, the

cytotoxicity of the copolymers, either complexed with DNA or in the free form,

is inversely proportional to the mol fraction of these comonomers Thisreduction is even more than what can be expected based on the DMAEMA mol

fraction in the copolymer (14 and Bos and Hennink, unpublished data)

NVP-DMAEMA copolymers synthesized by polymerization to high conversionshow excellent DNA binding, condensing characteristics and transfectioncapabilities This is ascribed to a synergistic effect of DMAEMA-richcopolymers and NVP-rich copolymers present in this system on the complex

formation with plasmid DNA (14).

Fig 1 Poly(2-[dimethylamino]ethyl methacrylate) (pDMAEMA)

Temperature-sensitive copolymers of DMAEMA and N-isopropylacryl

amide (NIPAAm) of various monomer ratios and molecular weights have

been evaluated as carrier systems for DNA delivery (16) All copolymers,

even with a low DMAEMA content of 15 mol%, were able to bind to DNA at

25°C Light-scattering measurements indicate that complexation is panied by precipitation of the copolymer in the complex caused by a drop ofthe lower critical solution temperature of the copolymer Thecopolymer:plasmid ratio, at which complexes with a size of approx 200 nmare formed, shows a positive correlation with the NIPAAm content of thecopolymer and is independent of molecular weight of the copolymer Com-plexes containing copolymers of low molecular weight or high NIPAAm con-tent prepared at 25°C aggregate rapidly when the temperature is raised to

accom-37°C On the other hand, complexes containing copolymers of high lar weight or lower NIPAAm content are stable at 37°C The cytotoxicity ofthe complexes decreases with increasing NIPAAm content and is indepen-dent of molecular weight of the copolymer The transfection efficiency as afunction of the copolymer:plasmid ratio shows a bell-shaped curve Thecopolymer:plasmid ratio at which the transfection efficiency is maximal riseswith increasing NIPAAm content; the maximum transfection efficiency drops

molecu-with increasing NIPAAm content of the copolymer (16).

Besides pDMAEMA, a number of structural analogs, differing in the side chain

groups, have been evaluated as transfectant (17,18) Almost all studied cationic

methacrylate/methacrylamide polymers are able to condense the structure ofpDNA, yielding small polyplexes (100–300 nm) and a slightly positive c potential.However, the transfection efficiency and the cytotoxicity of the polymers differwidely: the highest transfection efficiency and cytotoxicity are observed forpDMAEMA itself Assuming that polyplexes enter cells via endocytosis,pDMAEMA apparently has advantageous properties to escape the endosome A pos-

sible explanation is that, because of its average pK avalue of 7.5, pDMAEMA ispartially protonated at physiological pH and behaves as a proton sponge This mightcause a disruption of the endosome, which results in the release of both thepolyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol In con-

trast, the analogs of pDMAEMA studied have a higher average pK avalue andhave, consequently, a higher degree of protonation and a lower buffering capacity,which might be associated with a lower tendency to destabilize the endosome,

resulting in both lower transfection efficiency and a lower cytotoxicity (17,18).

Furthermore, structural analysis by molecular modeling techniques suggests that,

of all studied polymers, pDMAEMA has the lowest number of interactions withDNA The authors therefore hypothesize that the superior transfection efficiency

of pDMAEMA-containing polyplexes can be ascribed to an intrinsic property ofpDMAEMA to destabilize endosomes combined with an easy dissociation of the

polyplex once present in the cytosol and/or the nucleus (17,18).

Functionalization of pDMAEMA, e.g., by coupling of an antibody (-fragment)

or another ligand such as poly(ethylene glycol) is feasible by using a random

copolymers of DMAEMA with aminoethyl methacrylate (AEMA) (19) The

percentage of incorporated primary amino groups can be controlled by the feedratio of AEMA:DMAEMA, and is usually below 10 mol% The ligands can becoupled to the amine groups directly or, for example, via a thiol group In this case,following the synthesis of the copolymer, protected thiol groups are introduced in

a derivatization step with N-succinimidyl 3-(2-pyridyldithio) propionate and

subsequent treatment with dithiothreitol The obtained thiolatedp(DMAEMA-co-AEMA) can be conjugated to antibodies and other ligands, e.g.,the nuclear localization signal decapeptide Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Asp-NH2, via a disulfide linkage In general, the coupling efficiencies are high

(>90%) (19) The thiolated polymers can also be used to determine the apparent

kinetic rate constants between plasmid DNA and the nonviral carrier polymers

using surface plasmon resonance spectrometry (20) In this case, the polymers are

attached to the gold layer through the thiol groups

Freeze-drying of these gene delivery systems can be performed using a

controlled two-step drying process and sucrose as lyoprotectant (21)

Freeze-drying is shown to be an excellent method to preserve the size and transfection

potential of pDMAEMA–plasmid complexes (8,22), even after aging at 40°C

(23) The concentration of the sugars is an important factor affecting both the

size and transfection capability of the complexes after drying and thawing However, the type of lyoprotectant (sugar) used is of minor

freeze-importance (24) The DNA topology has been shown to affect

pDMAEMA-mediated 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 acombination of both methods for sterilization of pDMAEMA-based gene

transfer complexes have been assessed (26) Agarose gel electrophoresis and

circular dichroism spectroscopy shows that filtration of polyplexes does notchange the topology and integrity of the DNA Moreover, a full preservation ofthe transfection potential of the filtered polyplexes was observed Precoating

of the filter with polyplexes reduces the material loss and the loss oftransfectivity In contrast, autoclaving dramatically affects physical character-istics of polyplexes, resulting in a complete loss of transfection potential.Addition of sucrose to the preparation protects DNA present in pDMAEMA–DNA complexes, to some extent, from degradation during autoclaving, but thetransfection potential is not retained Filtration or autoclaving of polymer alonedoes not result in substantial loss of polymer integrity and material, or indecreased transfection potential Naked DNA can easily be sterilized byfiltration as well, although some DNA may be lost Therefore, separate

sterilization of polymer and DNA stock solutions, followed by asepticformation and handling of polyplexes, may be an acceptable and preferred

alternative to filtration of polyplexes (26).

In this chapter, protocols are provided for the synthesis of pDMAEMAhomo- and copolymers in water or toluene, gel permeation chromatogra-phy (GPC) analysis of the synthesized polymers, the preparation ofpDMAEMA–DNA polyplexes, and subsequent determination of the plas-mid integrity after formulation, sterilization of pDMAEMA-basedpolyplexes, a standard transfection protocol, and determination of cellviability and of transfection efficiency

2 Materials

2.1 Common

1 Water purified by reversed osmosis (RO water)

2 pH-meter or pH-indicator strips

3 70% Ethanol

4 Sodium chloride, pro analysi (pa).

5 Sodium hydroxide (4.0, 1.0, and 0.1 N).

6 Phosphate buffered saline (PBS): 3.6 mM KH2PO4, 6.4 mM Na2HPO4, and 145 mM

NaCl, pH 7.2

7 4-(2-Hydroxy-ethyl)-1-piperazine ethane sulfonic acid (HEPES)

8 0.22-µm Filters

9 Vortex mixer

10 Eppendorf tubes sterilized by autoclaving

11 Cell culture equipment: Biohazard safety cabinet or laminar airflow cabinet,equipped with burner and suction device, CO2 incubator 37°C, Bürker-Türk orBürker counting chamber (bright lined), microscope (e.g., ×25 objective, ×10ocular), water bath at 37°C, centrifuge

12 P-20, P-100, P-200, and P-1000 Gilson pipets with sterile tips

13 5- and 10-mL pipets

14 15- and 50-mL centrifuge tubes

2.2 Synthesis of pDMAEMA Homo- and Copolymers in Water

1 DMAEMA stabilized with 0.15% tert-butyl-hydroxytoluene (Fluka).

2 Ammonium peroxodisulphate (APS) (Fluka)

3 37% Hydrochloric acid, pa

4 100 mL Infusion bottles with plastic lids and silicon rubber septa (e.g., Emergo)

5 Needles

6 Nitrogen–vacuum exchange system

7 Water bath at 60°C

8 Vacuum distillation equipment

9 1–10 mL Glass syringes, glass pipets

10 Dialysis tubing, mol wt cut-off 12–14 kDa (e.g., Medicell)

11 Freeze-dryer

2.3 Synthesis of pDMAEMA in Toluene

1 DMAEMA stabilized with 0.15% tert-butyl-hydroxytoluene (Fluka).

2 _,_'-Azoisobutyronitril (AIBN) (Fluka)

10 Vacuum stove with temperature control at 40–60°C

11 Vacuum distillation equipment

12 Glass syringes, glass pipets

2.4 GPC of Water-Soluble pDMAEMA Homo- and Copolymers

1 Tris-(hydroxymethyl)-aminomethane (Tris)

2 NaNO3, pa

3 60% HNO3, pa

4 0.22-µm Filter (e.g., Millipore GVWP04700)

5 Degassing setup consisting of vacuum pump and ultrasound bath

6 High-performance liquid chromatography (HPLC) system, consisting of a pump,

an autoinjector, a refractive index detector, and software with GPC option andGPC column set option

7 GPC column set (e.g., Shodex K80P, KB-80M, and KB802); for molecularweight higher than 10,000 g/mol, the KB802 column can be skipped

8 1-mL Poly(propylene) (PP) shell vial with snap cap (Alltech)

9 2-mL Syringes

10 Nylon 13-mm syringe filters, 0.45 µm (Alltech)

11 15-mL PP tubes with cap

2.5 Amplification and Purification of Plasmid

1 Bacterial strain producing an appropriate marker gene

2 Plasmid purification kit (e.g., Giga-kit from Qiagen)

2.6 Preparation of pDMAEMA–DNA Polyplexes with Low DNA Concentration

1 Plasmid in TE-buffer (see Subheading 3.4.).

2 pDMAEMA (see Subheadings 3.1 and 3.2.).

2.7 Preparation of pDMAEMA–DNA Polyplexes with High DNA Concentration

1 Glacial acetic acid, pa

2 Sucrose

3 Plasmid in TE-buffer (see Subheading 3.4.).

4 pDMAEMA (see Subheadings 3.1 and 3.2.).

2.8 Integrity of Plasmid in Polyplexes

1 Pronase

2 Tris-(hydroxymethyl)-aminomethane (Tris)

3 Glacial acetic acid, pa

4 Ethylenediamine tetraacetic acid (EDTA)

5 Ethidium bromide (EtBr) or SYBR Green I nucleic acid stain (Molecular Probes)

6 Bromophenol blue

7 Glycerol

8 Marker DNA (e.g., h DNA [Gibco] digested with restriction enzyme PstI)

9 Polyaspartic acid (pAsp) (Sigma, mol wt 50,000)

10 Electrophoresis apparatus

11 UV detection system

12 Conical flask

13 Microwave oven

2.9 Sterilization of pDMAEMA-Based Gene Transfer Complexes

1 0.22-µm Filters (e.g., cellulose acetate) (Schleicher & Schull GmbH, Dassel,Germany)

2.10 Standard Transfection Protocol

1 OVCAR 3, COS 7 or other cells

2 Completed culture medium, depending on the particular cell line

benzene sulfonic acid hydrate (XTT) (Gibco)

2 N-methyl dibenzopyrazine methylsulfate (PMS) (Gibco).

15 5-Bromo-4-chloro-3-indolyl-`-D-galactopyranoside (X-Gal, Gibco).

16 Dimethylsulfoxide (DMSO): 99.9% spectrophotometric grade

3.1 Synthesis of pDMAEMA Homo- and Copolymers in Water

Synthesis of pDMAEMA (Fig 1) is achieved by radical polymerization (9,10),

and it can be performed in either an acidified aqueous solution (this procedure)

or in toluene (see Subheading 3.2.) Because the ester bond of DMAEMA can

be chemically hydrolyzed in water (27), synthesis of pDMAEMA in toluene may

be preferred However, pDMAEMA is routinely synthesized in water, which isespecially useful for the synthesis of copolymers of DMAEMA and toluene-insoluble monomers After synthesis, the polymers are characterized by nuclearmagnetic resonance (NMR) to determine if all monomer has reacted, and in thecase of copolymers, also the copolymer composition Furthermore, the polymersare characterized by GPC to determine the average molecular weight and the

molecular weight distribution (see Subheading 3.3.).

1 The monomer DMAEMA should be vacuum-distilled shortly before synthesis to

remove the radical scavenger tert-butylhydroxytoluene After distillation, allow

nitrogen into the distillation equipment In some cases, the comonomer should be

vacuum-distilled as well (see Note 1).

2 Dissolve 1.0 g initiator APS in 15 mL water in a 50-mL flask

3 An initial DMAEMA concentration of 20% (v/v) for the synthesis is recommended The molecular weight of the polymer will depend on the ratio of monomer and

initiator (M:I ratio) used Several typical examples of mixtures are given in Table 1.

Add in 100-mL infusion bottles with septum appropriate amounts of water,37% HCl and DMAEMA (in that order) Use a glass syringe to transferDMAEMA into the flask When copolymers are synthesized, the molar quanti-ties of DMAEMA and the comonomer should match the quantity of DMAEMA

given in the Table 1 Adjust the pH of the solution to 5.0 to prevent hydrolysis of

the esterbond of DMAEMA In this stage, chain transfer agents such asmercaptoethanol can be added to obtain low-molecular weight polymers with a

functional endgroup Add the appropriate amount of APS solution (see Table 1).