EVALUATION OF A NOVEL BIORESORBABLE POLYESTER AS
GENE DELIVERY CARRIER
Pablo C. Caracciolo1, Francisco Parra2, Gustavo A. Abraham1, Blanca Vázquez2, Julio San
Román2, Lucía L. Policastro3,4, Osvaldo L. Podhajcer3
1
Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA, UNMdP-CONICET), Av. J. B.
Justo 4302, Mar del Plata, Argentina.
2
Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), Juan de la Cierva 3, Madrid, España.
3
Laboratorio de Terapia Molecular y Celular, Fundación Instituto Leloir-CONICET, Av. Patricias Argentinas
435, Buenos Aires, Argentina.
4
Departamento de Micro y Nanotecnología, Comision Nacional de Energia Atómica (CNEA-CONICET), Av.
Gral. Paz 1499, San Martín, Buenos Aires, Argentina.
E-mail: pcaracciolo@fi.mdp.edu.ar
Abstract. In the last decades, gene therapy has gained attention due to its potential to treat chronic diseases and
genetic disorders, in addition to cancer and infectious diseases such as AIDS. Cationic polymers can potentially
complex DNA, not only protecting it from nuclease degradation, but also generating polyplexes with a nanoscale
size small enough to enter the cell through endocytosis. Polycations can provide a pH-buffering ability allowing
them to behave as “proton sponges”, assisting the escape of complexes from endosome and potentially
improving the transfection efficiency. In this work, a bioresorbable polymer potentially suitable for DNA
complexation and gene delivery was synthesized and studied as gene delivery carrier. A polyester based on
tetraethylene glycol (TEG) and L-glutamic acid was synthesized from piro L-glutamic acid (pGlu) by aminogroup protection with di-tert-butyl dicarbonate. After deprotection, the amino moieties were available to interact
with DNA. Number average molecular weight of the polyester resulted 2430 Da, with a polydispersity of 1.4.
The material displayed a pKa of 6.8, which lays between cytosomal and endosomal pH. This result indicates that
the buffering capacity of the cationic polymer could facilitate endosomal escape of polymer-DNA polyplexes by
osmolysis. The polyester showed complexation capacity towards pGL3-Promoter plasmid after assaying
different polymer/DNA ratios. Moreover, several concentrations of the polyplexes were tested against colorectal
cancer cells, displaying no cytotoxicity. The polyplexes, however, were not able to transfect cells, maybe due to
the low molecular weight of the polycation. In this direction, polyesters with the same backbone and higher
molecular weights are currently being synthesized to assess their transfection efficiency.
Keywords: Cationic
Transfection.
1.
polyester,
L-glutamic
acid,
DNA
complexation,
Cytotoxicity,
INTRODUCTION
In the last decades, gene therapy has gained attention due to its potential to treat
chronic diseases and genetic disorders, in addition to cancer and infectious diseases such as
AIDS. Despite the higher gene expression generally displayed by viral vectors, there are
concerns over the immunogenicity, low DNA cargo capacity, cytotoxicity, and
recombinogenic potential of viral gene transfer.
In recent years, there has been substantial progress in the development and application
of non-viral vectors [Liu et al., 2009; Jian et al., 2009; Mintzer et al., 2009]. Cationic
polymers can potentially complex DNA, not only protecting DNA from nuclease degradation,
but also generating polyplexes with a nanoscale size small enough to enter the cell through
endocytosis. Polycations could provide a pH-buffering ability allowing them to behave as
“proton sponges” [Behr, 1997; Sonawane et al., 2003; Satchi-Fainaro et al., 2006], assisting in
the escape of complexes from endosome and potentially improving the transfection
efficiency. Due to their improved safety profile, low immunogenicity and low cytotoxicity,
diverse non-viral vectors continue to be explored and optimized for gene delivery [Anderson
et al., 2004; Wolff, 2005]. However, there are still many problems to be solved before this
therapy becomes a standard clinical practice.
In this work, a bioresorbable polyester potentially suitable for DNA complexation and
gene delivery was synthesized employing tetraethylene glycol (TEG) and piro L-glutamic
acid (pGlu) and studied as gene delivery carrier. The polyester showed complexation capacity
towards pGL3-Promoter plasmid after assaying different polymer/DNA weight ratios. The
obtained complexes displayed no cytotoxicity against human colorectal cancer cells (LoVo).
Despite the buffering capacity of the synthesized polyester, and its ability to complex DNA,
the obtained polyplexes, however, were not able to transfect cells, maybe due to the low
molecular weight of the polycation. In this direction, polyesters with the same backbone and
higher molecular weights are currently being synthesized to assess their transfection
efficiency.
2.
MATERIALS AND METHODS
2.1
Synthesis of monomer pGluBOC-TEG-pGluBOC
pGlu and TEG were reacted at a molar ratio of 1:0.4 in refluxing toluene (20 % w/v)
for 12 h in the presence of p-toluene sulfonic acid (0.1:1 molar ratio to pGlu). Then, toluene
was distilled off and the oil was dissolved in dichloromethane (DCM, 20 % w/v) and washed
with an aqueous solution of saturated NaHCO3. The organic layer was dried and di-tert-butyl
dicarbonate, triethylamine and N,N-dimethylamino pyridine were added (2.4:3:0.1 molar
ratios to diester, respectively). The reaction was carried out at 25 °C for 24 h. Then, the
solution was washed with HCl 0.6 M. The organic layer was dried and purified by column
chromatography (DCM/methanol 35:1), obtaining a yellow oil (Yield: 78 %).
2.2
Synthesis of polyesters
Equimolar amounts of pGluBOC-TEG-pGluBOC and TEG were reacted in the
presence of NaCN (0.1:1 molar ratio to monomers) in N,N-dimethylformamyde (DMF, 300 %
w/v) at 25 °C for 24 h [Molina et al., 1993]. Then DMF was distilled off and deprotection of
the polymer was carried out in DCM:trifluoroacetic acid 2:1 (15 % w/v) at 25 °C for 1 h. The
polymers were purified by dialysis, and obtained as brown oils (Yield of BOC-protected
polyester: 72 %, yield of deprotected polyester: 67 %).
2.3
Physicochemical characterization
1
H, 13C, 1H-1H COSY, and 1H-13C HSQC NMR spectra were recorded on a
MERCURY-400 spectrophotometer. FTIR spectra were performed on a Perkin-Elmer
Spectrum One spectrophotometer equipped with an ATR system. High resolution mass
spectrometry was performed on an Agilent 6520 Accurate Mass Q-TOF LC/MS. Size
exclusion chromatography (SEC) analysis were carried out employing DMF with 0.1 % LiBr
as solvent at 70 °C, a flow rate of 0.3 mL/min and polymer solutions (3 mg/mL).
Thermogravimetric Analysis (TGA) was performed with a TGA Q500 (TA instruments)
thermobalance under nitrogen atmosphere at a heating rate of 10 ºC/min and a temperature
interval between 30 and 800 ºC. Differential Scanning Calorimetry (DSC) was carried out
with a Perkin Elmer DSC-8500. The dry samples (10 mg) were heated from -60 to 120 ºC at a
constant rate of 10 ºC/min. The glass transition temperature (Tg) was taken as the middleset of
the heat capacity transition. The pKa of the deprotected polyester was determined by acidbase titration of 50 mg of polymer solution in a 25 mL aqueous 0.1M NaCl with NaOH 0.1
M, employing an automatic 842 Titrando (Metrohn). The pKa value was calculated from the
averaged inflection of the titration curve.
2.4
Amplification and purification of plasmid
The pGL3-Promoter Vector plasmid (Promega) was amplified in Escherichia coli
DH5-α and purified in an anion-exchange resin (Qiagen). DNA concentration was determined
by ultraviolet (UV) absorbance at 260 nm, and DNA integrity was verified by electrophoresis
on 1% agarose gel employing Tris acetate-EDTA buffer (pH 8.0).
2.5
Preparation and characterization of polyester/DNA complex
The polyester/DNA complex was prepared at several weight ratios. Aliquots
containing 1 g DNA were added to solutions with different amounts of the deprotected
polyester (5, 10, 20 and 40 g). The mixtures were gently stirred to favor complex formation.
After 15 min at room temperature, solutions were seeded in agarose gel (1% agarose solution
in Tris acetate-EDTA buffer with ethidium bromide 0.5 g/ml). Each well was loaded with 15
l of polyester/DNA complex containing 1 g of DNA. Complex formation was confirmed
by electrophoresis, carried out for 60 minutes at 100 V.
2.6
Evaluation of cell viability
Evaluation of cytotoxicity was performed by the colorimetric 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Human colorectal cancer cells (LoVo)
were seeded in a 96-well plate (5 x 104 cells per well) in DMEM cell culture medium with
10% fetal bovine serum (FBS) at 37oC in 5% CO2 atmosphere. After 24 h cells were
incubated with different polyester/DNA weight ratios for 24 h. The complex solutions were
then removed and fresh medium containing MTT (5 mg/mL) was added. After 2 h the
medium was removed, and formazan crystals were dissolved in dimethyl sulfoxide (DMSO).
Absorbance was measured at 550 nm using a microplate reader. Relative viability of treated
cells was calculated respect to non-treated control cells (arbitrary defined as 100% viability).
2.7
Evaluation of transfection capacity
Subconfluent cultures of LoVo cells were employed to evaluate the transfection
efficiency of the polyester/pGL3-promoter plasmid complex. Cells were seeded in a 24-well
plate (5 x 104 cells per well) in DMEM cell culture medium with 10% fetal bovine serum
(FBS) and incubated for 24 h at 37oC in 5% CO2 atmosphere. Then, polyester/DNA complex
with different weight ratios was added to cells and incubated for 4 h at 37 oC, ensuring a load
of 1 g of DNA per well. The medium was replaced with fresh DMEM cell culture medium
and cells were incubated for 48 h and luciferase activity was evaluated. LipofectamineTM
2000 was used as control [Policastro et al., 2009].
3.
RESULTS AND DISCUSSION
3.1
Physicochemical characterization
H NMR spectrum of the monomer (Fig. 1A) showed signals for CO2CH2 (4H,  =
4.31) and NCO2C(CH3)3 (18H,  = 1.47), indicating the presence of ester bonds and BOC
protection, respectively. FTIR spectrum showed three peaks in the carbonyl region: 1789 cm -1
(lactam), 1745 cm-1 (ester), and 1714 cm-1 (BOC). C–O–C ester peaks (1188 cm-1 and 1145
cm-1), and C–O–C ether peaks (1046 cm-1 and 1021 cm-1) (Fig. 2A). The molecular weight of
the monomer was determined by mass spectrometry, resulting 616.29 Da (target difference:
4.52 ppm).
1
H NMR spectrum of the BOC-protected polyester (Fig. 1B) showed the duplication in
the integration of the signals corresponding to TEG respect to the peaks of the monomer, and
the appearance of a signal of NHCO2C(CH3)3 (1H,  = 5.26). Otherwise, the absence of the
NCO2C(CH3)3 and NHCO2C(CH3)3 signals in the spectrum of the deprotected polyester (Fig.
1C) respect to the BOC-protected polyester could be observed, as well as the appearance of a
signal of CHNH3+ (3H,  = 8.54).
FTIR spectrum of the BOC-protected polyester (Fig. 2B) showed an important
decrease in the peak at 1792 cm-1 respect to the monomer spectrum, due to the cyanide
catalyzed polymerization reaction of the lactam-carbonyls with the hydroxyl groups of TEG,
and the appearance of a band at 3353 cm-1, associated to the N-H. FTIR spectrum of the
deprotected polyester (Fig. 2C) showed the absence of the BOC-carbonyl signal (1734 cm-1)
respect to the BOC-protected polyester spectrum, and the appearance of a peak at 1672 cm-1,
associated to trifluoroacetate carbonyl, as well as a broad band centered in the methylene
peak, associated to the N-H of the protonated amine.
Number average molecular weights of the BOC-protected and deprotected polyesters
resulted 3180 Da and 2430 Da, respectively, with polydispersities of 1.6 and 1.4.
The TGA curve of the BOC-protected polyester showed a 5% of mass loss at 172.5
°C, and two peaks of maximum mass loss at 201.0 °C and 310.7 °C, while deprotected
polyester displayed a curve showing a 5% of mass loss at 99.6 °C, and three peaks of
maximum mass loss, at 153.7 °C, 215.4 °C and 311.8 °C. These results indicate that the
deprotected polyester is thermally more unstable that the BOC-protected polyester.
From DSC analysis Tg of BOC-protected polyester resulted -13.7 °C, while the value
for the deprotected polyester was -18.1 °C. The higher Tg value for the protected polyester can
be associated to its decreased chain movility due to the presence of the BOC-protecting
group. No melting transitions were observed. The materials are amorphous and rubbery at
room temperature.
From the titration curve of the deprotected polyester a pKa of 6.8 was obtained, which
is between cytosomal and endosomal pH. This result indicates that the buffering capacity of
the cationic polymer could potentially facilitate endosomal escape of polymer-DNA
polyplexes upon the decrease of the pH in the endosomes by osmolysis (“proton sponge
effect”).
1
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Figure 1: 1H NMR spectra of synthesized (A) pGluBOC-TEG-pGluBOC monomer, (B)
BOC-protected polyester, and (C) deprotected polyester.
Absorbance
A
B
C
3500 3000 2500
1800 1600 1400 1200 1000
800
600
-1
Wavenumber (cm )
Figure 2: ATR-FTIR spectra of (A) pGluBOC-TEG-pGluBOC monomer, (B) BOC-protected
polyester, and (C) deprotected polyester.
3.2
Characterization of polyester/DNA complex
In order to determine the optimal complexation conditions, we evaluated the degree of
binding between the deprotected polyester and the pGL3-Promoter plasmid at different
weight ratios. The obtained complexes were visualized by agarose gel electrophoresis. As
shown in Fig. 3, the polyester retarded DNA plasmid for all weight ratios, and the retardation
increased at higher ratios. This incomplete retardation could suggest a low charge density for
the polyester.
Weight ratio
(polyester/DNA)
0/1
5/1
10/1 20/1 40/1
Figure 3: Gel retardation assay. Polyester/DNA complex prepared at different weight ratios.
The cytotoxicity of the polyester/DNA complex was evaluated by the MTT assay
performed in LoVo cells. Figure 4 shows the effect of different polyester/DNA weight ratios
on cell viability, demonstrating that the complex is not toxic to cells in the studied range.
100
Cell viability(%)
80
60
40
20
0
0/1
5/1
10/1
20/1
40/1
Weight ratio (polyester/DNA)
Figure 4: Cytotoxicity assay. Polyester/DNA complex prepared at different weight ratios.
LoVo cells were also employed to assess the transfection efficiency of the polyester/
pGL3-Promoter plasmid complex at different weight ratios. Unfortunately, low levels of
luciferase activity were measured for all ratios after 48 h of incubation.
4.
CONCLUSIONS
In this work, we have obtained a bioresorbable cationic polyester based on L-glutamic
acid and tetraethylene glycol. The material resulted amorphous and rubbery at physiological
temperature. The polyester was able to perform DNA complexation due to its protonated
amino groups. Moreover, the obtained complexes displayed no cytotoxicity against human
colorectal cancer cells (LoVo). Despite the buffering capacity of the synthesized polyester,
and its ability to complex DNA, low transfection efficiency was obtained for polyplexes with
different weight ratios. These results can be attributed to the low molecular weight of the
polycation. In this direction, polyesters with the same backbone and higher molecular weights
are currently being synthesized to assess their transfection efficiency.
ACKNOWLEDGEMENTS
The authors thank M. Falciola for her technical support.
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