The use of folate-PEG-grafted-hybranched-PEI nonviral vector for the inhibition
of glioma growth in the rat
Bing Lianga,1, Ming-Liang Hec,1, Chu-yan Chanc, Yang-chao Chenc, Xiang-Ping Lid, Yi Lia, Dexian Zhengf,
Marie C. Line, Hsiang-Fu Kungc, Xin-Tao Shuaib,*, Ying Penga,**
aDepartment of Neurology, The Second Affiliated Hospital, Sun Yat-sen University, No. 107 West Road of Riverside, Guangzhou 510120, China
bBiomedical Engineering Center, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
cThe Center for Emerging Infectious, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong
dDepartment of Otolaryngology, Nanfang Hospital, Nanfang Medical University, Guangzhou, China
eDepartment of Chemistry, The University of Hong Kong, Hong Kong
fDepartment of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, China
a r t i c l e i n f o
Received 18 February 2009
Accepted 13 April 2009
Available online 8 May 2009
In vivo gene transfer
Combined gene therapy
a b s t r a c t
Combined treatment using nonviral agent-mediated enzyme/prodrug therapy and immunotherapy had
been proposed as a powerful alternative method of cancer therapy. The present study was aimed to
evaluate the cytotoxicity in vitro and the therapeutic efficacy in vivo when the cytosine deaminase/5-
fluorocytosine (CD/5-FC) and TNF-related apoptosis-inducing ligand (TRAIL) genes were jointly used
against rat C6 glioma cells. The potency of the FA-PEG-PEI used as a nonviral vector was tested in the FR-
expressed C6 glioma cells and Wistar rats. The C6 glioma cells and animal model were treated by the
combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL. The antitumor effect was eval-
uated by survival assays and tumor volume. This study revealed a significant increase of cytotoxicity
in vitro following the combined application of FA-PEG-PEI/pCD/5-FC and FA-PEG-PEI/pTRAIL treatments
in C6 glioma cells. Animal studies showed a significant growth inhibition of the C6 glioma xenografts
using the combined treatment. These results demonstrated that the combined treatment generated
additive cytotoxic effect in C6 glioma cells in both in vitro and in vivo conditions, and indicated that such
treatment method using both enzyme/prodrug therapy and TRAIL immunotherapy might be a promising
therapeutic strategy in treating glioma.
? 2009 Elsevier Ltd. All rights reserved.
Glioma is the most common primary malignancy of the brain. It
is known as a highly chemoresistant and radioresistant cancer with
high morbidity, mortality and extremely grim prognosis. The
median survival time of glioma patients is generally less than 2
years, despite multi-modality treatments with extensive surgical
resection, radiotherapy, chemotherapy or immunotherapy. Recent
advances in the neurosurgical technique, radiation therapy and
chemotherapy have all failed to improve the survival rate of this
group of patients . Therefore, novel strategy is urgently needed
for promoting the survival rate of the glioma patients. In recent
studies, gene therapy has been proposed as one of the potential
strategies which bears several advantages over conventional drug
therapies [2,3]. A long-term expression of high dosage therapeutic
could be delivered locally, and specifically to target tissues, which
reduce the risk of nonspecific toxicity and ineffective dosing. In this
study, the gene therapeutic strategies being investigated were
based on some previously established anti-neoplastic principles,
genes and immune-enhancing cytokine genes [3–6].
Among all gene therapeutic strategies now being investigated,
suicide gene/prodrug system has been recognized as one of the most
effective method in treating tumors. It has been revealed as a highly
potent agent in treating most chemoresistant and radioresistant
tumors [7–9]. One of the most widely investigated suicide gene/pro-
drug systems is the cytosine deaminase/5-fluorocytosine (CD/5-FC)
Abbreviations: CD, cytosine deaminase; FA, folate; FA-PEG-PEI, folate-PEG-
grafted-hyperbranched-PEI; FR, folate-receptor; pDNA, plasmid DNA; PEI, poly-
(ethylene imine); PEG, poly(ethylene glycol); TRAIL, TNF-related apoptosis-inducing
ligand; 5-FC, 5-fluorocytosine; 5-FU, 5-fluorouracil.
* Corresponding author. Tel.: þ86 20 8411 0365; fax: þ86 20 8411 2245.
** Corresponding author. Tel.: þ86 20 8133 2095; fax: þ86 20 8133 2833.
E-mail addresses: firstname.lastname@example.org (X.-T. Shuai), email@example.com
1These two authors contributed equally to the manuscript.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biomaterials
0142-9612/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved.
Biomaterials 30 (2009) 4014–4020
which has been studied extensively during the last decade. Cytosine
deaminase is an enzyme that could be found in bacteria and fungi. It
the highly cytotoxic 5-fluorouracil (5-FU). The latter compound then
metabolite to inhibit thymidylate synthase or to act as false bases in
DNA and RNA, thereby killing cells that are in the S-phase of the cell
cycle . By expressing the CD gene, and by administering water-
tumor cells. More importantly, 5-FC prodrug is membrane permeable
which has high bioavailability that could penetrate easily via the
‘localized’ 5-FU chemotherapy might therefore avoid the toxicity
associated with systemic 5-FU therapy, leading to a higher intra-
tumoral concentration. In addition, the characteristic that the 5-FU
a powerful bystander effect . This effect is essential in maximizing
only a small percentage of cells expresses CD.
Anothereffective antitumor strategy thatisproposed currently is
the apoptosis-inducing gene therapy. A wide variety of apoptosis-
inducing molecules have been identified to combat tumor cells.
Amongall, theligand-type cytokinemolecules of thetumornecrosis
factor (TNF)familyare recognized as the best candidate.TNF-related
apoptosis-inducing ligand (TRAIL) is a type II transmembrane
molecule, in which the carboxyl-terminus of the receptor-binding
domain protrudes extracellularly . Recombinant soluble human
TRAIL has already been employed in clinical investigation as cancer
therapy for it has been shown to induce apoptosis in various human
cancers. It functions by triggering the apoptotic signal cascades
through binding cognate receptors displayed on the cell surface. It
was also noted to exhibit potent antitumor activity without induced
toxicity in healthy tissue in various cancer xenograft models .
To date, the major challenge in gene therapy is to develop
a highly effective gene delivery system with low toxicity. Nonviral
vector is still an attractive option although the current agents being
used displayed disadvantages, e.g. low transfection efficiency and
high toxicity . To overcome the problem of high cationic toxicity
[i.e. polyethylene imine (PEI)] and low transfection efficiency [i.e.
PEGylated PEI (PEG-PEI)], our team has linked a cell specific tar-
geting molecule folate (FA) on polyethylene glycol (PEG). The FA-
PEG was then grafted onto the hyperbranched PEI (25 kD). Folate is
a common targeting ligand used for anti-cancer agents, since its
target (i.e. folate receptor) is often overexpressed in tumor cells (i.e.
C6 cell line) yet, rarely found in normal tissue, especially in the
normal brain tissue . Therefore, the FA has been used to test its
enhancing effect on vector delivery in FR-enriched tumor cells such
as C6 glioma cells . In our pervious study, the FA-PEG-grafted-
plasmid DNA (pDNA) into nanoparticles with a positive surface
charge under a suitable N/P ratio of 15 . In the present study, the
potency of the FA-PEG-PEI which could be used as a nonviral vector
was tested in the FR-expressed C6 glioma cells and Wistar rats.
could effectively condense
2. Materials and methods
2.1. Plasmids and chemicals
Plasmids pCMVCD was kindly provided by Dr. W. Walther (Max-Delbru ¨ck-
Center for Molecular Medicine, Berlin,Germany) and the pCMVTRAIL was producted
by our lab. Plasmid DNAs were amplified in Escherichia coli and were purified
according to the manufacturer’s instructions (QIAGEN, CA, USA). The quantity and
quality of the purified pDNA were assessed by measuring its optical density at
260 nm and 280 nm, and by electrophoresis in 1% argrose gel, respectively. The
purified plasmid DNAwas kept in aliquots at a concentration of 1 mg/ml. In this study,
all chemicals including PEI 25,000 Da, monomethoxy PEG (mPEG-OH) 3400 Da, 5-
fluorocytosine and (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) were products from Sigma–Aldrich (St Louis, MO, USA). Polyplexes, i.e. the
delivery agent/pDNA complexes, used throughout the present study were prepared
at N/P 15 according to our previous in vitro results that the polyplexes received the
highest transfection efficiency in C6 glioma cells whilst a low cytotoxicity at this N/P
2.2. Synthesis of delivery agents
a-Hydroxy-3-amino-poly(ethylene glycol) (HO-PEG-NH2) (Mn¼3.4 kDa, Mw/
Mn¼1.15) was prepared according to a report by Kataoka et al. . To conjugate
folate to HO-PEG-NH2, folic acid (2 mmol) was dissolved in anhydrous DMSO
(20 ml). N-hydroxysuccinimide (NHS, 4 mmol) and dicyclohexylcarbodiimide (DCC,
4 mmol) were added and the mixture was stirred overnight at room temperature.
The mixturewas mixed with a DMSO solution of HO-PEG-NH2, and TEA solution (pH
8.0) was added. The mixture was then filtrated, dialysized against deionized water
(MWCO: 1000 Da), and lyophilized. FA-PEG-OH thus prepared was converted into
FA-PEG-COOH by reaction with succinic anhydride (SA). FA-PEG-OH and SA (1:5 in
molar ratio) were dissolved in 20 ml anhydrous chloroform and refluxed at 70?C for
48 h. After chloroform was removed by distillation, polymer was re-dissolved in
20 ml deionized water and dialyzed against water for two days to remove small
molecular succinic acid and succinic anhydride. Polymer solution was freeze-dried
to yield pure FA-PEG-COOH. FA-PEG-PEI was synthesized and characterized as
previously described . In brief, FA-PEG-COOH (1 mmol) was activated with NHS
(2 mmol) for 24 h in dry dichloromethane (20 ml) containing dicyclohexyl-
carbodiimide (1.2 mmol) as a catalyst. The precipitated 1,3-dicyclohexylurea (DCU)
was removed by filtration. The filtrate was added to diethyl ether and cooled at 4?C
for 2 h. The precipitate was collected by filtration and dried under vacuum at room
temperature. Hyperbranched PEI 25 kDa and the NHS activated PEG were dissolved
in PBS (pH 7.4) and magnetically stirred for 24 h at room temperature to produce FA-
PEG-PEI. The mixture was purified by membrane dialyses (MWCO: 8000 Da) in
distilled water for 1 dayand the solution was lyophilized. Nontargeting PEG-PEI was
synthesized by the same approach using the NHS/DCC chemistry. PEG grafting
2.3. Cell culture
Glioma C6 cells were obtained from American Type Culture Collection (ATCC)
and maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penn/strep,
Invitrogen Corporation) in a humidified atmosphere of 5% CO2at 37?C. When the
cell confluence of 90% was reached, they were trypsinized and subcultured. All cell
culture reagents were purchased from Invitrogen Corporation (Carlsbad, CA, USA).
2.4. Western blotting analysis
The C6 glioma cells (1?105) were seeded in 24-well plate a day before trans-
fection. They were bathed in DMEM with 10% FBS complete media and were incu-
bated in a humidified atmosphere with 5% CO2at 37?C until the cell confluence was
around 70%. Four hours prior to transfection, the media was removed and replaced
with fresh DMEM with 10% FBS. To test the expression of plasmids, pCMVCD and
pCMVTRAIL,1 mg of DNA was diluted in 50 ml of serum-free DMEM in an Eppendorf
tube and mixed thoroughly. Based on an N/P ratio of 15, corresponding quantity of
FA-PEG-PEI was added to a 50 ml serum-free DMEM in another sterilized tube and
the sample was vortex mixed immediately. Both mixtures were then left to incubate
at room temperature for 5 min. Samples of both tubes were then vortex mixed
together and was left for incubation at room temperature for 30 min. The original
cell culture media was replaced with a 100 ml complex solution whilst a 200 ml
serum-free DMEM was added on top for each well. They were incubated at 37?C for
4 h. Thereafter, the transfection medium was changed with fresh and complete
DMEM culture media. At 72 h after the transfection, cells were washed twice with
PBS and lysed with SDS sample buffer. Protein samples (20 mg) were separated using
SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The
membranes were incubated at room temperature for 30 min in a blocking buffer (5%
low fat milk, 150 mm NaCl, and 20 mm Tris–HCl, pH 7.5), and were then incubated
with a sheep primary antibody (1:200 dilution) against the CD or a rabbit antibody
(1:500 dilution) against the TRAIL and caspase-3 (Covance, Richmond, CA, USA).
Secondary antibody, horseradish peroxidase (HRP)-conjugated anti-sheep or anti-
rabbit IgG, was used to amplify the signal. The blots were developed using chem-
iluminescence system (New Life Science Products, Boston, MA, USA) and the results
were photo-documented. The membranes were then washed again with buffer, and
were rehybridized with a primary antibody for b-actin (1:500 dilutions). HRP-
conjugated antibody and chemiluminescence system were used for the detection of
b-actin as described earlier. The proteolysis of caspase-3 was evaluated by Western
blotting by using antibodies against caspase-3 that could detect both unprocessed
proenzyme and active forms after cleavage.
B. Liang et al. / Biomaterials 30 (2009) 4014–40204015
2.5. In vitro 5-FC sensitivity
The C6 cells were grown in 96-well plates at an initial density of 6000 cells/well.
They were transfected with the same concentration as that used for the 24-well
plates. Transfection was performed by using 300 ng of pDNA in 150 ml of serum-free
growth medium. Four hours later, the transfection medium was changed with fresh
and complete DMEM culture media, and 5-FC (0–160 mg/ml) was added concur-
rently. After 72 h of incubation, 150 ml of serum-free growth medium was replaced,
and 20 ml of MTT solution (5 mg/ml) was added. The cells were then incubated for
around 4 h before a 100 ml of DMSO was added. After gentle agitation for 5 min, the
absorbance at 570 nm of each well was measured using the FLUOstar microplate UV
2.6. In vivo studies
C6 glioma cells were stereotactically implanted into the right caudate nucleus of
male Wistar rats (250–280 g). Briefly, rats were anesthetized with ketamine (20 mg/
kg) and placed in a stereotactic frame (Wood Dale, IL, USA). With a Hamilton syringe,
C6 glioma cells (5?105) in 20 ml serum-free DMEM were injected through a burr
hole into the right caudate nucleus (3 mm lateral and 1 mm anterior to the bregma,
5 mm deep from the dura) over 10 min. The syringe was then retracted over 5 min.
The burr hole was filled with bone wax.
Five days after the injection, the rats were divided into four groups (9 rats per
group) for immunocytochemical and survival rate analyses. Different injections
were carried out on the right striatum of each rat according to grouping. Details are
listed as follows: Group 1: PBS buffer (50 ml); Group 2: 40 mgof pCMVTRAIL /FA-PEG-
PEI (50 ml, N/P¼15); Group3: 40 mg of pCMVCD/FA-PEG-PEI (50 ml, N/P¼15); Group
4: 40 mg of pCMVTRAILþpCMVCD/FA-PEG-PEI (50 ml, N/P¼15); and four normal
rats were kept as control. The injectionwas performed at 5 different points along the
needle tract (6, 5.5, 5, 4.5, 4 mm deep from the dura), over a period of 10 min;
afterwards, the needle was left in place for 5 min and was then retracted over 5 min.
The burr hole was blocked with bone wax. Two days after injection, 5-FC was
administrated intraperitoneally at 250 mg/kg/day (the serum 5-FC consentration
might be 100 mg/ml) for 14 consecutive days .
All animals were kept under the same laboratory conditions with no steroids or
antibiotics. When symptoms including severe paresis and/or ataxia, or more than
20% of body weight were lost, the animal was sacrificed. The total volume of the
tumor (cubic millimeters) was calculated by summing up the cross-sectional areas.
Survival rate was analyzed by a log-rank test based on the Kaplan–Meier survival
analysis by using MedCalc statistical software.
2.7. Immunohistochemistry study
Rats were killed and perfused intracranial with warm normal saline (150 ml)
followed by 4% paraformaldehyde (150 ml). The brains were removed, and all
sections were cut at 3–5 mm thick from buffered formalin-fixed, paraffin-embedded
tissue. After deparaffinization, sections were stained with haematoxylin/eosin
(H&E). For histopathological analysis, at least five paraffin sections from each animal
were used for hematoxylin/eosin staining. Immunohistochemisty studies were
performed in serial sections obtained from the paraffin blocks which were meant for
histological diagnosis. They were immunostained with CD and TRAIL monoclonal
antibodies (dilution 1:500). The one step Envision polymer (Dako A/S, Glostrup,
Denmark) was used as a secondary link to DAB chromogen. Prior to the application
of the primary antibody, antigen retrieval was carried out by three-minute incu-
bation with a pressure boiler in citrate buffer solution (pH 6.0).
2.8. Statistical analysis
All data were analyzed with SPSS 13.0. The results are expressed as mean ?SE
and the statistical significance was defined as P<0.05.
3.1. Combination of CD/5-FC and TRAIL enhanced cytotoxicity
The antitumor effect of the combined treatment using CD/5-FC
and TRAIL was evaluated at various 5-FC concentrations. In the
study, C6 cells were transfected with FA-PEG-PEI/pCMVCD alone or
in combination with FA-PEG-PEI/pCMVTRAIL with various 5-FC
concentrations. As illustrated in Fig. 1, the viability of C6 cells was
94.2% when treated with FA-PEG-PEI/pCMVCD alone (i.e. without
5-FC). Such cell viability was similar to that when cells were
transfected with FA-PEG-PEI/pEGFP. Cell viability was significantly
lower (50.2%) for samples treated with the combined FA-PEG-PEI/
pCMVTRAIL agent. At a 5-FC concentration of 80 mg/ml, MTT assay
revealed a 42.5% cell viability for samples treated with FA-PEG-PEI/
pCMVCD alone while 23.4% for the combined treatment. Based on
the data, the combined treatment using CD/5-FC with TRAIL
produced significantly higher cytotoxicity than the case when they
were used as a single agent. When the 5-FC concentration reached
80 mg/ml or higher (i.e. 160 mg/ml), no significant difference was
found between the cytotoxicity induced in different treatment
groups. High cell viability constantly remained in the control cells,
which were transfected with FA-PEG-PEI/pEGFP, despite any vari-
ation in the 5-FC concentration. In general, the level of cytotoxicity
of the 5-FC was neglectable in the controls.
3.2. The expression of the CD, TRAIL and activated caspase-3
protein in FA-PEG-PEI/pDNA complexes transfected C6 glioma cells
In this study, C6 glioma cells were transfected with FA-PEG-PEI/
pCMVCD complexes at an N/P ratio of 15, and similar procedures
were carried out for FA-PEG-PEI/pCMVCD. The expression of the
CD, TRAIL and activated caspase-3 protein was examined using
Western blotting. In Fig. 2, the expression of CD and TRAIL proteins
as well as the caspase-3 activation in C6 cells after FA-PEG-PEI/
pCMVCD and FA-PEG-PEI/pCMVTRAIL transfection is shown. There
were obvious protein bands in the transfected cells and the acti-
vation of caspase-3 confirmed that the CD and TRAIL proteins had
been expressed and activated.
5-FC Concentrations (ug/ml)
Fig.1. The cell viability of the C6 glioma cells changed with various 5-FC concentrations (mean ?SD, n ¼ 4) after transfection of FA-PEG-PEI/pEGFP, FA-PEG-PEI/pCMVCD or FA-PEG-
B. Liang et al. / Biomaterials 30 (2009) 4014–40204016
3.3. Effect of combined therapy of CD/5-FC with TRAIL genes on C6
glioma cells in vivo
The rat C6 glioma models were employed in our study for
examining the combined treatment effect invivo. Three weeks after
the initial injection, all animals in the control group died due to
excessive tumor burden. The average tumor size for the PBS-control
groups was 172.52 ?8.02 mm3, while 53.13?3.72 mm3noted for
the combined therapy (i.e. CD/5-FC and TRAIL) group. For the
groups treated with CD/5-FC or TRAIL single gene, the average
respectively (Fig. 3). According to our results, the combined therapy
could significantly diminish the glioma tumor size when compared
with the controls or single gene therapy (p<0.01).
In this study, the survival time course of the tumor-bearing rats
was also recorded. As seen in Fig. 4, all Wistar rats in the control
groups died before day 20. Among the other three treatment
groups, rats belonged to the combined therapy group demon-
strated a longer survival time course when compared with the CD/
5-FC and TRAIL single therapy groups (p<0.01). No significant
statistical difference was found between the two single therapy
groups (p>0.05). On the 35th day after C6 glioma cells’ implan-
tation, about 2/3 of the CD/5-FC and TRAIL single treated rats
survived but the rest died in the next 15 days. For the combined
treatment group, 80% of the animals survived until day 35 and 2 of
them were still alive after 80 days of implantation.
During necropsy, tumors excised from the control group were
fairly large in size, and hypervascularized with petechia and central
necrosis. As shown in Fig. 5B, the histopathological analysis of
tumors excised from the control showed generally larger tumors
(6–8 mm) with scattered necrosis, haemorrhage with cerebral
edema (data not shown). Microscopic view showed that they were
mostly hypercellular with nuclear pleomorphism and scattered
haemorrhage (Fig. 6a(B)). Gliomas isolated from the combined
therapy group were generally small and pale with fewer visible
superficial blood vessels (Fig. 6a(E)). For rats that had a survival
time course longer than 60 days (i.e. from the combined therapy
group), the tumors were tiny like nodules in the brains. For those
two rats that survived up to 80 days, they were found tumor-free
whilst microscopic examination disclosed cyst formation with
macrophage infiltration (Fig. 6a(E)). These indicated that the
combined application of CD/5-FC and TRAIL was effective in sup-
pressing and treating glioma growth in vivo.
Immunohistochemisty studies were performed to confirm the
expression of the CD and TRAIL proteins in the rat brains. Negative
control is shown in Fig. 6b(A) andthe cells were stainedblue as well
as in the PBS-control group. Marked membranous CD and TRAIL
distribution with nuclear immunostaining was partly noted in the
CD/5-FC and TRAIL single treated rats (Fig. 6b(C, D). Diffuse, cyto-
plasmic and focally membranous distribution of CD and TRAIL
immunoreactivity was revealed by DAB immunostaining, as seen in
rats from the combined therapy group (Fig. 6b(E)).
Nowadays, no treatment is yet found to be effective in treating
malignant glioma. The use of conventional (i.e. chemotherapeutic)
agents is often limited by the systemic toxicity. Surgical removal of
tumor mass together with postoperative radiotherapy and/or
chemotherapy is the most common strategy applied in glioma
patients, but the survival rate is usually less than 2 years after diag-
have tumor recurs within 2 cm from the primary site . Recent
progress in the molecular and cellular biology has promoted gene
therapy as a promising treatment strategy in brain tumors .
Therapeuticvectorswhichcoulddirectly injectedintothe gliomavia
the conventional surgical approach . This is because lesser
Fig. 2. Western blot analysis of the expression of CD, TRAIL, and the caspase-3 acti-
vation in the C6 glioma cells after FA-PEG-PEI/pCMVCD or pCMVTRAIL transfection.
CD: cytosine deaminase; TRAIL: TNF-related apoptosis-inducing ligand; C/T: CD/TRAIL.
Tumor Size (mm3)
p < 0.01
p < 0.01
p < 0.01
0Control (PBS) CD/5-FC
Fig. 3. The average tumor size in different groups after C6 glioma cells’ implantation.
They include the PS-control, EGFP-control, CD/5-FC single therapy, TRAIL single
therapy, and combined therapy (i.e. CD/5-FC with TRAIL) groups.
Survival probability (%)
Fig. 4. The survival probability of the rats in different groups after C6 glioma cells
implantation. They include the PS-control, CD/5-FC single therapy, TRAIL single
therapy, and combined therapy (i.e. CD/5-FC with TRAIL) groups.
B. Liang et al. / Biomaterials 30 (2009) 4014–40204017
Fig. 5. The brain sections of the C6 glioma cell implanted rat brains. (A) PS-control, (B) CD/5-FC single therapy, (C) TRAIL single therapy, and (D) combined therapy (i.e. CD/5-FC with
Fig. 6. a: Histological characteristics of the C6 gliomas in the C6 glioma cell implanted rat brains (sections were stained with H&E). b: Immunohistological characteristics of the C6
gliomas in the C6 glioma cell implanted rat brains (sections were immunostained with CD and TRAIL monoclonal antibodies respectively). (A) Normal rat, (B) PS-control, (C) CD/5-FC
single therapy, (D) TRAIL single therapy, and (E) combined therapy (i.e. CD/5-FC with TRAIL) groups.
B. Liang et al. / Biomaterials 30 (2009) 4014–40204018
normal tissues would be affected in the new approach, while some
The suicide gene/prodrug treatment strategy is originally devel-
oped based on the concept of the chemotherapeutic agent-induced
systemic toxicity. It was postulated that the overall toxicity might be
minimized if the tumor is targeted correctly or the surrounding
cellular enzymes are capable to convert nontoxic prodrugs to toxic
agents. In this study, the CD/5-FC system makes use of the CD gene
which could encode for an enzyme that works to convert 5-FC pro-
drug to 5-FU, thus results in tumor cell death and strong bystander
effect . This approach produces intense toxicity exclusively in the
vicinity of tumor, which could maximize the therapeutic efficacy.
novel combinations that could work well with other therapies are
still being investigated [9,23,24].
In previous studies, researchers had tried to improve the thera-
peutic efficacy of CD/5-FC by combining other genetic agents
including IL2andTRAIL [13,25], inwhich inspiring results have been
of the C6 cells to 5-FC by introducing TRAIL. A number of recent
studies have demonstrated that the combined treatment with TRAIL
and 5-FU could lead to increased suppression and regression of
tumor growth [13,26]. The cross-sensitization betweenTRAIL and5-
FU could induce apoptotic pathway through caspase activation,
which was found to depend on the expression of proapoptotic bax
chemotherapeutic agents could induce upregulation of death
receptor 5 (DR5) expression . Therefore, TRAIL application
together with the CD/5-FC system might be an effective therapeutic
strategy for C6 gliomas.
The development of a highly efficient, safe and cost-effective
vector is the major challenge of gene therapy. Although viral vectors
risk of immunogenicity, tumorigenicity and cytotoxicity .
Nonviral vectors which bear comparatively lower gene transfection
efficiency became a better option for they induce lesser immune
reaction and thus, are more cost-effective. Moreover, they have the
capability to deal with large DNA plasmids. They are easy to prepare
and flexible to use. Finally, the cell-type specificity which they
possess after chemical conjugation of a targeting ligand are all
advantages over the viral vectors . In our study, the high toxicity
of PEI and the low transfection efficiency of PEG-PEI have been
overcome by the production of FA-PEG-PEI for gene delivery. Such
vector has the characteristics of good biocompatibility, potential
biodegradability, and relatively high gene transfection efficiency.
Folate as a targeting ligand was ligated on the vector, since folate
receptors (FR) were generally overexpressed in human cancer cells.
Enhanced expression of human FR has also been reported in malig-
While in normal human tissues, FR has a limited expression mainly
found in kidney, lung, choroid plexus, and placenta .
Initially, transfection efficacy of FA-PEG-PEI mediated delivery of
ratio ranged from 5 to 30; data not shown). An N/P ratio of 15 was
different cell lines including HEK 293T, glioma C6 and hepatoma
HepG2 cells . The advantages of 5-FC/CD gene therapy had been
described. First, the mechanism of the antitumor effect was inde-
pendent of the cell cycle. Second, the prodrug 5-FC was highly
permeable and readily crossed the BBB. Cerebrospinal fluid distri-
bution from the blood sera of 5-FC was w60–80%. Drug delivery to
the tumor was one of the most important factors for successful
chemotherapy and could be the largest factor limiting clinical
strong and did not require direct cell-to-cell contact . The
system to sustain effective killing of glioma cells. Therefore, the C6
cells were transfected with FA-PEG-PEI/pCMVCD alone at an N/P
ratio of 15 whilst the5-FC concentrationwasvariedaccordingly. Our
results indicate that theoptimal concentrationof 5-FCformaximum
cytotoxicity to generate was 80 mg/ml, which agreed with previous
report . The combined transfection of FA-PEG-PEI/pCMVCD with
FA-PEG-PEI/pCMVTRAILattheN/Pratio of 15 wasalsotestedagainst
various 5-FC concentrations. As seen in Fig. 1, combined therapy
generated additive cytotoxic effect in the C6 cells than when the
agent was applied alone invitro. The dose of i.p. 5-FC for invivo gene
therapy ranges from 250 to 500 mg/kg/day, and which was about
100–200 mg/ml the achieved dose of 5-FC in the serum of injected
Inordertoinvestigate the underlyingmechanism of the additive
cytotoxic effect, the expression of CD and TRAIL proteins were
analyzed using Western blotting. The level of activation of caspase-
3 was also examined, for it is a good indicator of apoptotic level in
mammalian cells. In a recent study, TRAIL was found to induce
apoptosis via interaction with DR5, which was mediated by cas-
pase-3-initiated pathways in sensitive glioma cells . In our
study, both the CD and TRAIL proteins have demonstrated their
antitumor efficacy through the apoptotic process as shown in Fig. 2.
The synergistic antitumor effects performed by the combined CD/
5-FC and TRAIL proteins/prodrug might partly, if not all, were
related to the activation of apoptotic process in the glioma samples.
On the other hand, in vivo study has demonstrated a significant
delay in human tumor growth with the combined gene therapeutic
approach. The results were relatively correlated with the in vitro
condition, which indicate a delay in tumor cell proliferation (i.e.
MTT assay) and acceleration in apoptosis. The combined FA-PEG-
PEI/pCMVCD/5-FC and pCMVTRAIL treatment has revealed signif-
icant tumor suppression in Wistar rats by decreasing the tumor
volume and increasing the survival rate of the animal models.
These results suggested that the combined treatment strategy
using FA-PEG-PEI/pCMVCD/5-FC and FA-PEG-PEI/pCMVTRAIL was
an effective strategy that deserved further investigation in both
in vitro and in vivo conditions.
The present study demonstrated an effective antitumor effect
induced by CD gene transfected TRAIL gene, which was mediated
by the FA-PEG-PEI. Combined treatment with CD/5-FC and TRAIL
had synergistic inhibitive ability, when compared with the single-
gene therapy with either CD or TRAIL gene. Our data suggested that
an effective antitumor efficacy might be achieved by using FA-PEG-
PEI vector bearing the CD and TRAIL genes. This combined treat-
ment might be a treatment strategy that would help promote the
survival rate of glioma patients. Nevertheless, further investiga-
tions and more in vivo studies would be required to confirm its
optimal dosage and safety in clinical application.
This research work was supported by the 863 Programs of China
(2007AA021101 to YP) and the National Science Foundation of
China (NSFC) (30672411 to YP and 50673103 and 50830107 to XS),
the Ph.D. Programs Foundation of Ministry of Education of China
(20050558084 to YP and 20060558083 to XS) and Research Grant
Council (RGC) of Hong Kong (CUHK7394/04M, to MLH). Thanks to
Dr. W. Walther (Max-Delbru ¨ck-Center for Molecular Medicine,
Berlin, Germany) for kindly providing the plasmids pCMVCD. There
are no potential conflicts of interest.
B. Liang et al. / Biomaterials 30 (2009) 4014–4020 4019
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