Biodegradable polyester, poly[α-(4-Aminobutyl)-L-glycolic acid], as a non-toxic gene carrier

Article abstract of DOI:10.1023/A:1007552007765

Purpose. The aim of this study was to develop a non-toxic polymeric gene carrier. For this purpose, biodegradable cationic polymer, poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA) was synthesized. PAGA was designed to have ester linkage because polyesters usually show biodegradability. Methods. Degradation of PAGA in an aqueous solution was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PAGA/DNA complexes were characterized by gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS). The transfection was measured by using the β-galactosidase reporter gene. Results. PAGA was degraded in aqueous solution very quickly and the final degradation product was a monomer (L-oxylysine). Formation of self-assembling biodegradable complexes between PAGA and DNA at a charge ratio 1:1 (+/-) was confirmed by gel band shift assay and AFM. In these studies, controlled release of DNA from the complexes could be seen. The complexes showed about 2-fold higher transfection efficiency than DNA complexes of poly-L-lysine (PLL), a structural analogue of PAGA, which is the most commonly used poly-cation for gene delivery. The polymer did not show cytotoxicity, possibly because of its degradability and the biocompatibility of the monomer. Conclusions. The use of the biodegradable poly-cation, PAGA, as a DNA condensing agent will be useful in safe gene delivery.

Full text of DOI:10.1023/A:1007552007765

Pharmaceutical Research, Vol. 17, No. 7, 2000
Research Paper
(11,12). However, it is degraded slowly and is toxic as shown
in cell culture studies (13,14). Although biodegradable polymer
was used previously as a nanoparticle DNA carrier (15), a
soluble and charged biodegradable polymer has not yet been
used as a gene carrier.
Biodegradable Polyester, Poly[␣-(4-
Aminobutyl)- -Glycolic Acid], as a
L
Non-Toxic Gene Carrier
We synthesized a novel PLL analogue, PAGA, containing
both a degradable ester linkage and a positively charged back-
bone. The polymer was degraded very quickly in aqueous solu-
tion. This polymer possessed the capability of condensing DNA
into compact complexes and showed fast hydrolytic degradation
after cellular uptake. Furthermore, PAGA/DNA complexes
exhibited higher transfection efficiency in cells than did PLL/
DNA complexes. The PAGA also showed much less toxicity
Yong-Beom Lim,1 Sang-Oh Han,1 Han-Uk Kong,1
Yan Lee,1 Jong-Sang Park,1 Byeongmoon Jeong,2
and Sung Wan Kim2,3
Received January 7, 2000; accepted April 11, 2000
Purpose. The aim of this study was to develop a non-toxic polymeric than did the PLL. Therefore, the newly designed PAGA can
gene carrier. For this purpose, biodegradable cationic polymer, poly[␣-
(4-aminobutyl)- -glycolic acid] (PAGA) was synthesized. PAGA was
designed to have ester linkage because polyesters usually show
biodegradability.
be substituted for PLL gene carriers and other positively charged
polymers giving increased transfection and non-toxicity.
L
MATERIALS AND METHODS
Materials
Methods. Degradation of PAGA in an aqueous solution was followed
by matrix-assisted laser desorption/ionization time-of-flight mass spec-
trometry (MALDI-TOF MS). PAGA/DNA complexes were character-
ized by gel electrophoresis, atomic force microscopy (AFM), dynamic
light scattering (DLS). The transfection was measured by using the ␤-
galactosidase reporter gene.
Results. PAGA was degraded in aqueous solution very quickly and
the final degradation product was a monomer (L-oxylysine). Formation
of self-assembling biodegradable complexes between PAGA and DNA
at a charge ratio 1:1 (ϩ/Ϫ) was confirmed by gel band shift assay and
AFM. In these studies, controlled release of DNA from the complexes
could be seen. The complexes showed about 2-fold higher transfection
PLL (mol. wt. 4,000) and 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma (St. Louis, MO). pSV-␤-gal plasmid vector (6,821 bp)
from Promega (Madison, WI). The matrix, ␣-CHCA was from
Aldrich (Milwaukee, WI) and used without further purification.
PAGA Degradation Study by Matrix-Assisted Laser
Desorption/Ionization Time-of-flight Mass
Spectrometry (MALDI-TOF MS)
efficiency than DNA complexes of poly-L-lysine (PLL), a structural
analogue of PAGA, which is the most commonly used poly-cation for
gene delivery. The polymer did not show cytotoxicity, possibly because
of its degradability and the biocompatibility of the monomer.
Conclusions. The use of the biodegradable poly-cation, PAGA, as a
DNA condensing agent will be useful in safe gene delivery.
PAGA was dissolved in 25 mM Hepes, pH ϭ 7.3 at a
concentration of 5 mg/ml and was incubated at 37ЊC. ␣-cyano-
4-hydroxy cinnamic acid (␣CHCA) was used as the matrix,
which was prepared at a concentration of 10 mg/ml in water/3%
TFA/acetonitrile, 4:1:6 (v/v). At an appropriate time interval, a
1 ␮l aliquot of PAGA solution was added to the 9 ␮l of the
matrix solution in a microcentrifuge tube. One ␮l aliquot was
applied to the MALDI sample plate and dried in vacuum. A
N2 laser radiating at 337 nm wavelength with 3 ns pulses was
used in a Voyager Biospectrometry Workstation (Perseptive
Biosystems, Framingham, MA). The ions generated by the laser
pulses were accelerated to 29 kV energy in a positive mode.
All the spectra were obtained at the same laser power. Molecular
weight (Mp, the most probable peak molecular weight deter-
mined from the highest peak) of PAGA determined was 3,200.
KEY WORDS: PAGA; gene delivery; biodegradable; ester linkage;
non-toxic gene carrier; controlled release.
INTRODUCTION
In recent years, gene therapy has become an important
area, as a new therapeutic method (1,2). Several human clinical
trials are under way to treat various diseases utilizing viral
vector gene carriers (3). Viral vectors include adenoviruses and
retroviruses that demonstrated high transfection efficiency but
are limited due to adverse effects such as immunogenicity,
toxicity, and mutagenesis caused by the cell-infected viruses
(4). Synthetic gene carriers, poly-cationic polymers (5–9) or
cationic liposomes (10) form complexes with plasmid DNA
or antisense oligonucleotide, which then can be carried into
cell compartments.
Gel Band Shift Assay
PAGA/DNA complexes were formed by mixing pSV-␤-
gal plasmid solution (20 ␮g/ml in H2O) with an equal volume
of PAGA in Hepes buffered saline (15 mM Hepes, 150 mM
NaCl, pH 7.3) (HBS) at an appropriate charge ratio. After
allowing 1 h for the complexes formation, samples were electro-
phoresed through a 0.8 % agarose gel at 70 V for 40 min and
stained with ethidium bromide to visualize the DNA.
PLL is the polymer most frequently used as a gene carrier
1 School of Chemistry & Molecular Engineering, Seoul National Uni-
versity, Seoul 151-742, Korea.
2 Center for Controlled Chemical Delivery (CCCD), Department of
Pharmaceutics and Pharmaceutical Chemistry, University of Utah,
BPRB, Room 205, Salt Lake City, Utah 84112.
Atomic Force Microscopy
3 To whom all correspondence should be addressed. (e-mail:
rburns@pharm.utah.edu)
DNA (pSV-␤-gal) was dissolved in Hepes-Mg (25 mM
Hepes, 10 mM MgCl2, pH 7.6) buffer (16) at a concentration
811
0724-8741/00/0700-0811$18.00/0 ᭧ 2000 Plenum Publishing Corporation

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Lim et al.
of 1 ␮g/ml to obtain a DNA image. The 2 ␮l of the solution
was deposited onto a freshly cleaved mica substrate. The solu-
tion was allowed to adsorb for 2 min, washed with 1 ml of
distilled water, and rapidly dried in a stream of N2 gas. For
PAGA/DNA complexes and the degraded complexes images,
complexes with 5:1 (ϩ/Ϫ) charge ratio were made by mixing
pSV-␤-gal plasmid solution (5 ␮g/ml in H2O) with an equal
volume of PAGA solution in H2O. The complexes solution was
incubated at 37ЊC to allow the degradation of the complexes
to occur. At an appropriate time interval, 2 ␮l of the complexes
solution was deposited onto a freshly cleaved mica substrate.
The solution was allowed to dry for 2 min and then excess
fluid was wicked off with filter paper. The solution was dried
at room temperature before imaging. AFM was performed using
a Nanoscope IIIa equipped with a E scanner (Digital Instru-
ments, Santa Barbara, CA). All AFM imaging was conventional
ambient tapping mode AFM with scan speeds of about 5 Hz
and data collection at 512 ϫ 512 pixels.
Fig. 1. The synthetic scheme of PAGA and the structure of PLL.
The monomer was polymerized by melting condensation as
shown in Fig. 1. Any side products or products from the poly-
merization should not be toxic to cells. The difference between
PAGA and PLL is only in the backbone linkage, an ester bond
in PAGA versus an amide bond in PLL.
Dynamic Light Scattering and Zeta Potential
The Z-average particle size and polydispersity index of
the PAGA/DNA complexes were determined by DLS at 25ЊC
with a Malvern 4700 system using a 25 mW He-Ne laser
(␭ ϭ 633 nm) as the incident beam at a scattering angle of 90Њ
and the Automeasure version 3.2 software (Malvern Instrument
Ltd, UK). For data analysis, the viscosity (0.8905 mPa.s) and
refractive index (1.333) of pure water at 25ЊC were used. PAGA/
DNA complexes were prepared as described above.
Degradation of PAGA in an Aqueous Solution
Molecular weight and degradation of PAGA in an aqueous
solution were investigated by using MALDI-TOF MS (Fig. 2).
It was shown that Mp, the most probable peak molecular weight
determined from the highest peak from the MALDI-TOF MS
spectrum, could be used to estimate the molecular weight of
polymer (18). So we followed Mp change of PAGA from the
MALDI-TOF MS spectra for degradation study. The degrada-
tion of PAGA was characterized by rapid initial degradation
within 100 min and gradual degradation until it is degraded to
Transfection and Cytotoxicity Assay
293 cells were seeded at a density of 6 ϫ 104 cells/well
in 24-well plate, 24 h prior to transfection. Due to its rapid
degradation in aqueous conditions, PAGA was dissolved in
water just prior to mixing with plasmid DNA. Plasmid pSV-
␤-gal/PAGA complexes were prepared by mixing pSV-␤-gal
(10 ␮g/ml) and PAGA (100 ␮g/ml) in an FBS-free cell culture
medium at an appropriate charge ratio, and incubated for 20
min at room temperature to achieve complexes formation. The
medium in the 24-well plate was replaced with transfection
mixture followed by 4 h of incubation at 37ЊC. Then the trans-
fection mixture was replaced with fresh medium. Cells were
further incubated for 48 h at 37ЊC. The ␤-galactosidase activity
in transfected cells was determined using a colorimetric ONPG
(o-nitrophenyl ␤-D-galactopyranoside) assay system (Promega,
Madison, WI). Absorbance at 405 nm was normalized to the
protein concentration determined using the Pierce BCA protein
assay reagent (Pierce, Rockford, IL).
monomer (L-oxylysine). The fast degradation of PAGA seems
to come mainly from self-degradation via hydrolysis of it own
ester linkage by the ε-amino groups. PAGA degraded com-
pletely to monomer within 6 month at 37ЊC. The degradation
study was also performed on PLL under the same conditions
For the cytotoxicity assay, 293 cells were treated with
PAGA or PLL in a 96-well plate, followed by 24 h incubation
at 37ЊC. Then the cytotoxicity was measured by MTT assay
(17). Briefly, 25 ␮l of the 5 mg/ml stock solution of MTT were
added to each well, and after 2 h of incubation at 37ЊC, 100
␮l of the extraction buffer (20% w/v of SDS in a solution of
50% of DMF, pH 4.7) were added. After an overnight incubation
at 37ЊC, absorbance at 570 nm was measured.
RESULTS
Synthesis of PAGA
Fig. 2. Degradation study of PAGA in aqueous solution by MALDI-
TOF MS. Mp: the most probable peak molecular weight determined
from the highest peak. Mp can be interpreted as a molecular weight
For the synthesis of the PLL analogue having an ester
backbone, a new monomer, L-oxylysine, was made from L-
lysine by converting the ␣-amino group into a hydroxyl group. of PAGA. The spectrum was the sum of 128 laser shots.

Biodegradable PAGA as a Non-Toxic Gene Carrier
813
or in a pseudo-extracellular milieu. Even after 3 months, PLL Size Distribution of the Complexes by Dynamic Light
hardly degraded (data not shown).
Scattering
Size distribution of PAGA/DNA complexes measured by
dynamic light scattering shows that the complexes had average
size of 326 nm at a charge ratio of 5:1 (ϩ/Ϫ) (Fig. 5). The
size distribution was rather heterogeneous as demonstrated by
the polydispersity index (0.252). A tendency for the complexes
size to increase was found as the charge ratio (ϩ/Ϫ) was
increased (data not shown).
Gel Band Shift Assay
The DNA condensing ability of PAGA was determined
by gel band shift assay. The positively charged PAGA makes
strong complexes with the negatively charged phosphate back-
bone of DNA. When the charge ratio (ϩ/Ϫ) reaches 1:1, no
free DNA is in solution (Fig. 3a). Since the degradation of
PAGA is fast, as shown in Fig. 2, it is crucial to verify the
stability of the PAGA/DNA complexes over the time needed
for transfection. Stability studies were done by measuring disso-
ciated DNA, in the form of a band, from the complexes at
pH ϭ 7.3 and 37ЊC (Fig. 3b–d). We observed slower degrada-
tion rate of the complexes than PAGA alone. The complexes
dissociated completely within one day, while PLL/DNA com-
plexes were stable for four days (Fig. 3e).
Transfection and Cytotoxicity
The transfection efficiency of PAGA was investigated and
compared with its structural analogue, PLL. The transfection
efficiency was measured using colorimetric ␤-galactosidase
enzyme activity assay (ONPG assay). The transfection protocol
was optimized for each of the polymers and done in the presence
of chloroquine (100 ␮M). It was found that optimum transfec-
tion of PAGA and PLL was obtained at charge ratios (ϩ/Ϫ)
of 40:1 and 5:1, respectively. The transfection efficiency of
PAGA/DNA complexes on 293 cells under optimized condition
was nearly twice that of PLL/DNA complexes as shown in
Fig. 6.
Atomic Force Microscopic Image of PAGA/DNA
Complexes During Degradation
All the possible forms of plasmid DNA, super-coiled,
nicked circular, and linear plasmid DNA can be seen in the
AFM image (Fig. 4a). Mg2ϩ was used to obtain a plasmid DNA
image because DNA binding to mica has been shown to be
enhanced in the presence of divalent metal counterions (19).
But in obtaining PAGA/DNA complexes images, a divalent
cation was not needed. Formation of self-assembling complexes
between PAGA and DNA can be seen in Fig. 4b at charge
ratio (ϩ/Ϫ) of 5:1. The shapes of the complexes were largely
spherical but rather heterogeneous. The toroidal shape can be
seen in Fig. 4b. The sizes of the complexes range from 100 to
400 nm. After incubation of the complexes at 37ЊC for 4 h,
degradation was apparent (Fig. 4c). A protruding DNA strand
The toxicity of PAGA on the cells was assayed using a
MTT assay (Fig. 7). After incubating 293 cells for 24 h with
100 ␮g/ml PAGA, the cells showed neither a decrease in popula-
tion nor a change in morphology indicative of minimal toxicity.
In contrast, PLL exhibited much increased toxicity when com-
pared to PAGA (about 25%). The PLL treated cells granulated,
and the cell population decreased under the same concentrations
as PAGA. At higher concentrations of PAGA, up to 300 ␮g/ml,
there was no significant cytotoxicity observed (data not shown).
DISCUSSION
The present article deals with the use of a novel biodegrad-
from the complexes could be seen with the shape of the com- able polymer as a gene carrier. The design principle that we
plexes becoming homogeneous and globular. After 8 h incuba- used for the synthesis of PAGA was that the polymer should
tion, more of the DNA was released from the complexes (Fig. be biodegradable. Currently, most non-viral gene carriers are
4d). Two apparent characteristics of 24 h incubation images subdivided into cationic polymers and lipids. Most of them
were that most of the DNA was released from the complexes have amide bonds (e.g., PLL) or vinyl bonds (e.g., poly(ethyl-
and that the density of the complexes was significantly reduced eneimine)). These bonds are very stable in aqueous solution
(Fig. 4e). From gel band shift assay, PAGA lost its ability to and there is no direct evidence of their degradation in body
make the complexes when the complexes were incubated for fluid. They are known to be cytotoxic (20). Also, most of the
24 h at 37ЊC (Fig. 3).
lipids used as gene carriers are known to be cytotoxic to some
Fig. 3. Formation of PAGA/DNA complexes and the degradation of the complexes. (A)
The effect of increasing proportions of PAGA on plasmid DNA (pSV-␤-gal) electrophoretic
migration; (B) The complexes were incubated at 37ЊC for 4 h; (C) 8 h; (D) 24 h. The
charge ratio between PAGA (ϩ) and DNA (Ϫ) is shown in each lane. (E) The degradation
study of the PLL/DNA complexes at the same condition as PAGA/DNA complexes. The
molecular weight of PLL (about 3500) used in this and the other experiment was similar
to the PAGA, and it was determined by MALDI-TOF MS. Samples were electrophoresed
through 0.8% agarose gel and stained with ethidium bromide to visualize DNA.

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Lim et al.
Fig. 4. AFM images. (A) plasmid DNA (pSV-␤-gal); (B) PAGA/DNA complexes; (C) after 4 h incubation; (D) after 8 h
incubation; (E) after 24 h incubation.
degree. To make non-toxic gene carrier, we designed the poly-
mer with a hydrolytically labile ester bond. Degraded monomer
or small oligomers will not elicit an immune response either.
The primary design of PAGA was from PLL. The amide back-
bone linkage of PLL was converted into ester linkage to confer
biodegradability upon PAGA.
Fig. 6. Transfection of PAGA/pSV-␤-gal or PLL/ pSV-␤-gal com-
plexes on 293 cells. Transfection efficiency was measured using colori-
metric ␤-galactosidase enzyme activity assay (ONPG assay).
Transfection protocol was optimized for each of the polymer and done
in the presence of chloroquine (100 ␮M). The data are expressed as
mean values (ϮSD) of 5 experiments.
Fig. 5. Dynamic light scattering of PAGA/DNA complexes. Size distri-
bution of PAGA/DNA complexes was measured by DLS. The com-
plexes were formed at 5:1 charge ratio (ϩ/Ϫ).

Biodegradable PAGA as a Non-Toxic Gene Carrier
815
solution. The dense core is likely to be DNA condensed by
PAGA. The dense core became more homogeneous than that
of intact complexes as the time progressed. This seems to occur
because the aggregated complexes were disaggregated as a
result of PAGA degradation. Near complete decomplexation
and a noticeable decrease in the complexes density after 24 h
indicated that almost the entire DNA had been released from
the complexes. Mica treated with Mg2ϩ containing buffer or
3-aminopropyltriethoxysilane (APTES) treated mica (AP-mica)
(16) is usually used in obtaining AFM images of DNA. Without
them, homogeneous spreading of DNA over a mica surface is
technically very difficult because DNA gathers together in a
very small region. The decrease in complexes density after 24
h seemed to result from the fact that the released DNA gathered
together in some very small region making them invisible in
the AFM image because the mica used for this experiment
was not treated with Mg2ϩ or APTES. From these sets of
experiments, we could conclude that PAGA/DNA complexes
are stable up to 24 h in an aqueous solution. When we think
that transfection mixtures are usually incubated for 4 h with
cells for in vitro transfection experiment, 24 h stability is suffi-
cient for transfection.
The transfection efficiency of PAGA/DNA complexes on
293 cells under optimized conditions was nearly twice that of
PLL/DNA complexes. This result may be explained by the
accelerated degradation of PAGA in cytoplasm and nucleus,
which enhances the expression of free DNA. Higher polymer/
DNA charge ratio for PAGA than PLL required for the optimum
transfection could also be explained by the degradation of
PAGA, which might demand more PAGA for DNA complex-
ation and negatively charged cell surface binding.
PAGA did not display any detectable cytotoxicity up to
high concentrations (300 ␮g/ml). But PLL had a noticeable
cytotoxicity in a concentration of 100 ␮g/ml. The toxicity of
cationic polymers has been a concern in their use as gene
carriers. Also, there is a notice of greater toxicity for some of
polymers applied in the absence of DNA (24). In this regard,
PAGA’s lack of toxicity without DNA complexation is a remark-
able finding. To our knowledge, there has been no gene carrier
that does not display any noticeable cytotoxicity without DNA
complexation. The degradable nature of PAGA, with its non-
toxic and biocompatible monomer and small oligomers, might
be important contributory factor to the non-toxicity of this
polymer. The non-toxicity of PAGA will be beneficial for in
vivo gene therapy.
In conclusion, the newly synthesized PAGA has the follow-
ing characteristics. First, the polymer has an overall positive
charge that forms stable complexes with DNA. Second, the
polymer shows accelerated degradation when free and
decreased degradation when it forms complexes with DNA.
Third, the final degradation product is a degraded monomer,
Fig. 7. Cytotoxicity of PAGA and PLL. Cell viability was measured
by MTT assay after 24 hr incubation of each polymer (100 ␮g/ml)
with the cells. The viability of cells that was not treated with polymer
was taken as 100%. The data are expressed as mean values (ϮSD) of
5 experiments.
The degradation study we performed in an aqueous solu-
tion demonstrates that PAGA is degraded rapidly within 100
min to small oligomers, and then to monomer in a gradual
manner. So, we could postulate that degraded PAGA would not
have an adverse effect on cell viability and it really didn’t
(vide ante). It seems that degraded fragments and monomer are
biocompatible. In addition, the small size of degraded oligomers
and monomer will not elicit complement activation that can be
a problem when larger ligand-DNA constructs are used (21).
The formation of condensed DNA by PAGA was investi-
gated using several techniques. In gel band shift assay, PAGA
was able to retard DNA migration as the charge ratio (ϩ/Ϫ)
increased. From this result, it was found that formation of the
complexes occurred at a near stoichiometric charge ratio
(ϩ/Ϫ). In the AFM image, the complexes were rather heteroge-
neous with some large particles around 400 nm. The overall
shape of the complexes was microaggregates. Experimental
data show certain condensed structures such as toroids and rods
can be induced by polyamines in DNA solutions ϳ1–10 ␮g/
ml (22). We could also see toroidal shaped complex with the
DNA concentration we used (5 ␮g/ml). The average size of
the complexes, 326 nm as determined by DLS, is in coherence
with that of complexes in the AFM image.
L
-oxylysine. Forth, the polymer condenses DNA into a spherical
Although PLL/DNA complexes were not decomplexed for
several days, PAGA/DNA complexes were gradually decom-
plexed to release free DNA from the complexes as shown in
gel band shift assay. This result indicates that by virtue of
degradation, PAGA lose its ability to condense DNA. But almost
all the DNA was in a condensed state for over 8 hr. Since only
a few hours are required for the maximum transfection of cells
(23), PAGA/DNA complexes can be utilized as a stable complex
system through cellular uptake. The stability of the complexes
shaped polymer. Fifth, the transfection efficiency of PAGA/
DNA complexes is about twice that of PLL/DNA complexes,
which possibly arise from the non-toxicity and degradability
of the polymer. Sixth, PAGA is non-toxic. We hypothesized
the faster degradation of PAGA will increase the release of free
DNA to the cytoplasm and /or the nucleus resulting in more
efficient expression of the transfected DNA. The degraded
PAGA oligomers or monomers will be rapidly removed from
investigated by AFM showed protruding DNA strands from the cellular compartments followed by metabolism and excretion
dense core when the complexes were incubated in an aqueous from the body. PAGA is a promising candidate as a gene carrier

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Lim et al.
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PAGA
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ACKNOWLEDGMENTS
We acknowledge support of this work from the Brain
Korea 21 program, Korea Research Foundation, Korea Science
and Engineering Foundation, and Expression Genetics Inc.
15. A. Maruyama, T. Ishihara, J. Kim, S. W. Kim, and T. Akaike.
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