Development of a safe gene delivery system using biodegradable polymer, poly[α-(4-aminobutyl)-L-glycolic acid] [19]

Full text of DOI:10.1021/ja001033h

6524
J. Am. Chem. Soc. 2000, 122, 6524-6525
Development of a Safe Gene Delivery System Using
Biodegradable Polymer,
Poly[r-(4-aminobutyl)-L-glycolic acid]
Yong-beom Lim,† Chang-hwan Kim,† Kwan Kim,†
Sung Wan Kim,‡ and Jong-sang Park*,†
School of Chemistry & Molecular Engineering
Seoul National UniVersity, Seoul 151-742, Korea
Center for Controlled Chemical DeliVery (CCCD)
UniVersity of Utah, Salt Lake City, Utah 84112
ReceiVed March 24, 2000
In recent years, there has been considerable interest in
biodegradable polymers that can be used as biomedical materi-
als.1,2 Much progress has been made in the field of drug delivery
systems in which many polymeric materials such as poly(lactide-
co-glycolide) showed promising properties as a controlled drug
reservoir.3 But the utilization of biodegradable cationic polymers
as nonviral gene carriers has been limited. Backbone linkages of
most current gene carriers are composed of amide or vinyl bonds,
which hardly degrade spontaneously in an aqueous solution. For
this reason, the potential for current nonbiodegradable nonviral
gene carriers to accumulate in an endosomal compartment or cell
nucleus and adversely interact with the host gene exists.4,5 These
issues present a problem with regard to using them to treat human
disease with gene therapy. Our ongoing research is focused on
the development of biodegradable and nontoxic cationic polymers,
the first of which was achieved by synthesizing poly(4-hydrox-
L-proline ester).6 The polymer showed relatively weak binding
ability to DNA, but cationic polymers binding strongly to anionic
DNA are required to condense DNA into a geometrically compact
shape. Here we report the synthesis of a novel biodegradable
cationic polymer, poly[R-(4-aminobutyl)-L-glycolic acid] (PAGA),
which forms DNA complexes strongly below a charge ratio of
2(().
Figure 1. Degradation profiles of PAGA incubated at 37 °C, pH 7.3.
Aliquots were taken at appropriate time intervals and MALDI spectra
were measured. Mp at 0 min (black line), 3300; 30 min (red line), 1600;
1 h (blue line), 1330; 3 h (yellow line), 1100; 5 h (violet line), 1050; 3
days (orange line), 990; 6 months (green line), NA. (NA ) not applicable).
Scheme 1. Synthetic Scheme of PAGA
First, monomer 1 (Nꢀ-cbz-L-oxylysine) was prepared by
converting an R-amino group of Nꢀ-cbz-L-lysine into a hydroxyl
group (Scheme 1). Polymerization of the monomer was conducted
in the melt to make polymer 2. The number- and weight-average
degrees of polymerization determined by gel permeation chro-
matography were 14 and 32, respectively. The polydispersity
index (2.3) obtained was rather broad, but reasonable as the
polymer was synthesized in the melt. Cbz groups protecting
ꢀ-amines were removed and the resulting primary amine groups
of PAGA were obtained as a hydrochloride salt. The molecular
weight distribution (MWD) of PAGA was determined by matrix-
assisted laser desorption/ionization time-of-flight mass spectrom-
etry (MALDI-TOF MS) as the MWD of charged, water soluble,
and fast degrading polymer is very difficult to measure by other
methods.7 The molecular weight of PAGA represented as an Mp
(the most probable peak molecular weight determined from the
highest peak intensity in the MALDI spectrum)6 was 3300 (Figure
1), which is an appropriate size for DNA condensation.
The structure of PAGA is similar to one of the polyamino acids,
poly-L-lysine (PLL), except for the backbone linkages. The
backbone linkages of PAGA and PLL are ester and peptide bonds,
respectively. PLL has been one of the most intensively studied
gene delivery carriers.8 Although peptide linkage is a natural bond
found in proteins and L-lysine, a monomeric unit of PLL, is an
amino acid, a homopolymer of L-lysine is not found in nature.
As a result, PLL is known to have cytotoxicity9 that possibly
arises from its slow degradation in vivo. We thought that the ester
backbone linkages in PAGA would make this polymer spontane-
ously degradable in an aqueous solution.
As a model of biodegradation, we incubated PAGA at 37 °C,
pH 7.3, and aliquots taken at appropriate time intervals were
subjected to MALDI-TOF MS. From the MALDI spectra shown
in Figure 1, it is evident that the degradation of PAGA occurred
very quickly in an aqueous solution. It needs only 30 min for Mp
for the intact polymer (0 min) to halve. When the time reached
5 h, Mp had reached 1050, which is a third of the polymer intact.
Fast degradation of PAGA implies that the polymer is probably
another example of “a self-destroying polymer” in which the main
chain cleavages occur by the nucleophilic attack of amine groups
of the polymer itself or of the nearby polymer molecules.6,10 The
degradation slowed after 1 day passed. Taking into account the
* To whom correspondence should be addressed.
† Seoul National University.
‡ University of Utah.
(1) Yasugi, K.; Nakamura, T.; Nagasaki, Y.; Kato, M.; Kataoka, K.
Macromolecules 1999, 32, 8024-8032.
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10.1021/ja001033h CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/23/2000

Communications to the Editor
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6525
Figure 2. (a) Fluorescence intensity change of DNA intercalated with
ethidium bromide. Mean ( standard deviation (n ) 3). (b) Absorbance
change profiles of DNA against nuclease (DNase I) attack. DNA alone
(dashed line), PAGA/DNA complexes at a charge ratio (() of 3 (dotted
line) and 5 (solid line). Absorbance was measured at 260 nm.
Figure 3. (a) Cytotoxicity assay of PAGA and PLL. (b) Transfection
efficiency in 293 cells. PLL with molecular weight of 4000 was used for
the experiments. Mean ( standard deviation (n ) 5).
a 35% increase in cell viability was observed after PAGA
treatment. The reason is unclear at this time, but it is certain that
PAGA is nontoxic to cells primarily due to its rapid biodegrada-
tion. On the other hand, PLL showed significant cytotoxicity in
that only 25% of the cells remain viable relative to nontreated
cells.
formation of amide bonds after the nucleophilic attack of ꢀ-amines
on backbone ester bonds, a slower degradation after 1 day can
be rationalized. The final degradation product of the polymer was
a degraded monomer (L-oxylysine) and required approximately
6 months.
The transfection efficiency of PAGA was compared with that
of PLL. Chloroquine, known to disrupt the membrane of the
endocytic vesicle, was used in this experiment. Optimal charge
ratios showing the highest transfection efficiency were different
between the two polymers, 6 and 60 for PLL and PAGA,
respectively. Charge ratios for complex formation were similar
between PLL and PAGA, between 1 and 2. But the 10-fold charge
ratio required for PAGA relative to that of PLL for the highest
transfection seems to result from the fact that degradation of
PAGA requires more of the polymer for PAGA/DNA complexes
to resist the harsh cellular environment. The transfection efficiency
of PAGA was about 3-fold higher than that of PLL under
optimized transfection conditions. Although small, we ascribe this
enhanced transfection efficiency of PAGA/DNA complexes to
the nontoxicity of PAGA and faster release of DNA from the
complexes due to degradation of PAGA after internalization
within cells or nuclei.
In conclusion, we have successfully developed a nontoxic
polymeric gene delivery system by using a biodegradable cationic
polymer, PAGA. The results indicate that the polymer degraded
quickly and completely in an aqueous medium as its backbone
linkages are composed of ester bonds. The formation of strong
interpolyelectrolyte complexes between the polymer and DNA
was observed and the polymer serves to protect DNA from
nuclease attack, an example of a harsh cellular environment.
Although the instability in aqueous conditions is a weakness of
PAGA in the present state, the finding that a biodegradable
polymeric gene carrier is nontoxic and shows enhanced trans-
fection is an important step toward making a safe gene carrier.
Intercalated ethidium bromide between the base pairs of DNA
is excluded when the interpolyelectrolyte complexes of polymer
and DNA formed.11 The fluorescence quenching which occurred
for a given concentration of DNA segments exposed to increasing
concentrations of PAGA is shown in Figure 2a as a function of
the positive to negative charge ratio. A sharp decrease in
fluorescence intensity occurred when the charge ratio (() reached
0.4, and it leveled off at a charge ratio of about 2. This result is
an indication of the strong binding of PAGA to DNA molecules
and the formation of PAGA/DNA complexes below a charge ratio
of 2.
For the DNA complexes of polymer to express the encoded
gene in the nucleus of cells, the polymer should protect DNA for
a few hours from nuclease attack in the cellular environment.12
Fragmentation of DNA was found to be fast as manifested by a
sudden increase in absorbance at 260 nm, occurring within 1 min
after addition of nuclease into DNA solution (Figure 2b). But
the DNA was partially protected from nuclease attack at a charge
ratio of 3 and complete protection was achieved at a charge ratio
of 5. The charge ratio (5, () required to protect DNA completely
is higher than is needed to form complexes and can be rationalized
by the degradation of PAGA. The protection continued throughout
an incubation of 4 h with nuclease, implying that the degradation
of DNA-complexed PAGA is not so fast compared to free PAGA.
We think that this is a result of the masking of the nucleophilic
primary amine groups of PAGA by DNA phosphates.
To verify that PAGA is not cytotoxic, the influence of PAGA
on cell viability was tested using a tetrazolium-based MTT
colorimetric assay (Figure 3a). As we expected, PAGA did not
display any detectable cytotoxicity even at high concentrations
(300 µg/mL). The cytotoxicity of cationic polymer/DNA com-
plexes is generally lower than that of the polymer alone.13 In
accordance with this, PAGA/DNA complexes did not show
cytotoxicity up to high charge ratios (60, (). Interestingly, about
Acknowledgment. We acknowledge the support of this work from
the Brain Korea 21 program, Korea Research Foundation, and Korea
Science and Engineering Foundation.
Supporting Information Available: Synthetic details, experimental
protocols, and MALDI spectrum (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
(11) Gershon, H.; Ghirlando, R.; Guttman, S. B.; Minsky, A. Biochemistry
1993, 32, 7143-7151.
(12) Ledley, F. D.; Ledley, T. S. AdV. Drug Del. ReV. 1998, 30, 133-150.
(13) Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova,
O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123-2133.
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