1904 Bikram et al.
Macromolecules, Vol. 37, No. 5, 2004
Baker (Phillipsburg, NJ ). N-Hydroxisuccinimide (NHS) was
purchased from Fluka (Milwaukee, WI). H-Lys(Z-OH) (cbz-L-
lysine) and N,N-dimethyl-His-OH (His) were purchased from
Bachem Bioscience Inc. (King of Prussia, PA). Poly-L-lysine
(PLL Mw 25 600) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-
tetrazolium bromide (MTT) were purchased from Sigma
Chemical Co. (St. Louos, MO). Ethylenediamine (EDA) was
purchased from Fisher Scientific (Fair Lawn, NJ ). Polystyrene
standards purchased from Polysciences, Inc. (Warrington, PA).
Dulbecco’s Modified Eagle Medium (DMEM), Dulbecco’s phos-
phate-buffered saline (PBS) and trypsin-EDTA were pur-
chased from Gibco BRL (Gaithersburg, MD). Heat-inactivated
fetal bovine serum (FBS) was purchased from Hyclone (Logan,
UT). Endofree Maxi Plasmid Purification kit was purchased
from QIAGEN (Valencia, CA). Luciferase assay system was
purchased from Promega Corp. (Madison, WI). Fluorescent
activated cell sorting (FACs) was performed using a FACScan
flow cytometer, Becton Dickinson (Palo Alto, CA). Propidium
iodide (PI) [2,7-diamino-9-phenyl-10-(diethyl aminopropyl)-
phenanthridinium iodide methiodide] was purchased from
Molecular Probes (Eugene, OR). Bicinchoninic acid (BCA)
reagent was purchased from Pierce (Rockford, IL). Protein
quantitation was determined with a Bio-Rad model 3550
Microplate reader from Bio-Rad Laboratories (Hercules, CA).
1H NMR spectra were obtained using a Varian Mercury 400,
Inc. (Palo Alto, CA) and chemical shifts (δ) were reported in
parts per million (ppm). RQ1 RNase-free DNase I enzymes
were purchased from Promega Corp. (Madison, WI). PELCO
mica disks, 9.9 mm diameter were purchased from Ted Pella,
Inc. (Redding, CA). Matrix assisted laser desorption ionization-
time-of-flight (MALDI-TOF) data were obtained using a
Voyager-DE STR Biospectrometry Workstation from Applied
Biosystems (Foster City, CA). Fourier Transform-Infrared (FT-
IR) spectra were obtained using a Mattson Galaxy Series 3000
FT-IR from Mattson Instruments Inc. (Madison, WI). The
molecular weights of the conjugates were determined by high
performance liquid chromatography - size exclusion chroma-
tography (HPLC-SEC) on Shodex OHpak (SB-806M HQ and
SB-803 HQ) columns in series using a Shidmadzu system and
a Shimadzu RID-10A refractive index detector purchased from
Shimadzu (Columbia, MD). pH measurements were deter-
mined with a Corning pH meter 340 purchased from Corning
Incorporated (Corning, NY). Atomic Force Microscopy (AFM)
pictures obtained with a Digital Instruments Nanoscope II
SFM model AFM (Santa Barbara, CA).
In the past, poly-L-lysine (PLL) has been widely used
in the field of gene delivery because its structure
facilitates various modifications of the polymer includ-
ing conjugation with ligands such as transferrin, epi-
dermal growth factor (EGF), and fusogenic peptides.13-15
However, the polycationic nature of PLL can affect
complex solubility at low N/P ratios (polymer nitrogens:
plasmid DNA phosphates), cell viability at high molec-
ular weights, and can lead to serum protein binding to
the polymer-DNA complexes. Previously, poly(ethylene
glycol) (PEG) has been used in combination with cationic
polymers such as PLL for use in gene delivery to
increase the pharmacokinetic properties of the polymer-
DNA complexes and reduce polymer toxicity.16-21 PEG
is a hydrophilic, nonionic or neutral polyether whose
steric stabilization properties impart high solubility,
nontoxicity, and nonimmunogenicity to molecules to
which PEGs are attached.22
Therefore, in an effort to develop a biocompatible,
cationic polymer with high transfection efficiency for use
in gene delivery, we have designed a novel water-
soluble, high molecular weight, biodegradable multi-
block copolymer (MBC) of the type (AB)n consisting of
repeating blocks of low molecular weight PEG attached
to low molecular weight PLL that was synthesized by
ring-opening polymerization of lysine N-carboxyanhy-
dride (NCA) to produce MBCs (PEG-PLL)x. In addition
to the steric stabilization properties of PEG, the use of
homobifunctional PEG in these MBCs has also facili-
tated the introduction of the biodegradable ester bond
functionalities into the backbone of the MBCs thereby
conferring degradability to the polymers.
In addition to reducing cellular cytotoxicities, the
biodegradable polymer also has to be able to maintain
high transfection efficiencies. However, even though
PLL has been widely used as a DNA-condensing poly-
cation, it produces low transfection efficiencies since it
cannot facilitate endosomal escape of the polymer/pDNA
complexes. This is because the pK of the primary amines
of PLL is above physiological pH whereas the endosomal
environment is acidic. Histidine and poly(L-histidine)
have both been shown to induce membrane fusion and/
or result in increased transfection efficiency of the gene
delivery system when conjugated to cationic poly-
mers.23-25 Furthermore, it has been reported that
R-amino groups on histidine residues result in elevation
of imidazole pK that can favor early endocytic release
of complexes.26 Consequently, the histidine derivative
N,N-dimethylhistidine was conjugated in various mole
ratios to the primary ꢀ-amines of PLL to produce the
final PEG-PLL-grafted-His (PEG-PLL-g-His) MBCs in
an effort to improve the endosomal escape of the
complexes after endocytosis.
Cell Lin es. The murine smooth muscle cell (A7r5) line was
purchased from American Type Culture Collection (ATCC)
(Manassas, VA) and was cultured in DMEM medium supple-
mented with 10% FBS and maintained at 37 °C in a humidified
atmosphere of 5% CO2.
Am p lifica tion a n d P u r ifica tion of p SV-EGF P a n d
p SV-Lu c. The pSV-EGFP vector was constructed by sub-
cloning the cassette for the simian virus 40 (SV40) promoter
and enhanced green fluorescent protein (EGFP) cDNA ob-
tained from the pCMS-EGFP vector (Clontech) into the pT/
BH vector that was a kind donation from Dr. Perry B. Hackett
(University of Minnesota). The pGL3-Promoter (pSV-Luc)
vector was purchased from Promega (Madison, WI). The
plasmids were amplified via transformation into J M109
competent cells and purified using a QIAGEN Endofree Maxi
Plasmid Purification kit.
Exp er im en ta l P r oced u r es
P olym er Syn th esis. The PEG diol was first converted to
a succinic diacid that was subsequently converted to the more
reactive homobifunctional NHS ester for nucleophilic substitu-
tion with the terminal R-amino group of PLL synthesized in
situ to produce the (PEG-PLL)x MBCs (Scheme 1).
Ma ter ia ls. All reactions were performed under an atmo-
sphere of dry nitrogen unless otherwise stated. Poly(ethylene
glycol) (PEG, Mw 1450) was purchased from Union Carbide
Corp. (Danbury, CT). Anhydrous methylene chloride (CH2Cl2),
succinic anhydride, 4-dimethylamino puridine (DMAP), 1,3-
dicyclohexylcarbodiimide (DCC), triphosgene, tetrahydrofuran
(THF), hexanes, sodium lump, dimethylformamide (DMF),
palladium, 10 wt % on activated carbon (Pd/C), formic acid,
dimethyl sulfoxide (DMSO), sodium deuterioxide solution
(DNaO), deuterium chloride solution (CID), trifluoroacetic acid
(TFA), benzene, Celite, acetone, and ethyl ether (ether) were
purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI).
Triethylamine (TEA) and dioxane were purchased from J . T.
Syn th esis of P EG Dia cid . PEG diol was dried by dissolv-
ing 25 g (17.24 mmol) of polymer in 20 mL of CH2Cl2 with
stirring. The polymer was precipitated by pouring the concen-
trated solution into 500 mL anhydrous ether at 4 °C with
vigorous stirring and the precipitate was then dried in vacuo.27
FT-IR (in CH2Cl2): broad O-H stretch: polymeric structures,
3333 cm-1; C-H stretch: methylene, 2840 cm-1, C-O-C
stretch: ether, 1100 cm-1. The number-average molecular
1
weight (Mn) determined by MALDI-TOF was 1435. H NMR