Carbohydrate Polymers 183 (2018) 29–36

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Research paper

The development of an alginate/polycaprolactone composite scaffold for in T

situ transfection application
⁎
Wei-Wen Hua,b, , Yun-Chung Wua, Zhe-Chen Hua
a
Department of Chemical and Materials Engineering, National Central University, Zhongli District, Taoyuan City 32001, Taiwan
b
Centre for Biomedical Cell Engineering, National Central University, Zhongli District, Taoyuan City 32001, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: Alginate and polycaprolactone (PCL) were coelectrospun using a dual-jet system to prepare composite nanofi-
Coelectrospinning bers in defined ratios, and hence both chemical properties and hydrophobicity of scaffolds can be manipulated.
Alginate These nanofibers were applied in gene immobilization: positively charged polyethyleneimine (PEI)/DNA poly-
In situ transfection plexes were adsorbed onto anionic alginate fibers, and the higher ratios of alginate resulted in the more im-
Composite nanofibers
mobilized nonviral vectors. Through the incorporation of PCL, biocompatibility of scaffolds was highly im-
Biocompatibility
proved. Finally, these scaffolds were used for in situ transfection application. Compared to pure alginate fibers,
composite fibers not only successfully transferred target genes to adhered cells but also enhanced cell mor-
phology and viability, suggesting that alginate/PCL nanofibers were multifunctional with gene delivery cap-
ability and biocompatibility, and the manipulation of their composition can balance and optimize both re-
quirements. To our knowledge, this approach might be the first one using electrostatic interactions to immobilize
genes onto nanofibrous scaffolds for in situ transfection application.

1. Introduction
In regenerative medicine, biological signals play critical roles be-
cause they can direct cell differentiation and promote appropriate tissue
regeneration. Protein-based bioactive factors are the most frequently
used ones. However, they are unstable in vivo due to enzymatic in-
activation. (Lee, Silva, & Mooney, 2011) In addition, the dosage need
for clinical purposes is extremely high cost. (Vo, Kasper, & Mikos, 2012)
In order to address these difficulties, regenerative gene therapy is de-
veloped as an alternative approach. (Munoz Ruiz & Regueiro, 2012)
Through gene delivery, transfected cells can continuously express
therapeutic genes in a sustained manner as small reactors. (Hu, Wang,
Hollister, & Krebsbach, 2007; Hu, Ward, Wang, & Krebsbach, 2010; Hu,
Wang, & Krebsbach, 2016) However, anionic nucleic acid molecules are
difficult to directly penetrate phospholipid-constructed cell membranes.
To overcome these barriers, polycations are broadly applied as nonviral
vectors. They can complex anionic DNA through electrostatic interac-
tions, and the formed nanoparticles exhibit positive charges which can
adsorb to negative-charged cell surfaces and facilitate endocytosis. For
example, polyethyleneimine (PEI) has been used to deliver os-
teoinductive genes to improve bone growth in skull defects, which
suggests the potential of using nonviral vectors to promote tissue re-
generation. (Huang, Simmons, Kaigler, Rice, & Mooney, 2005)
Although regenerative gene therapy can promote tissue repair, de-
livery of these therapeutic genes has been a challenge. If these nonviral
vectors are directly injected or inhaled, they may diffuse from target
sites immediately and lead to a systemic infection as well as serious
immune responses. (Chertok, Langer, & Anderson, 2016; Crystal, 1995)
To avoid unwanted side effects, substrate-mediated gene delivery is
considered as an attractive strategy for spatial control. Through im-
mobilizing gene vehicles on biomaterial surfaces, transfection can be
specific to the implant sites. (Jang, Houchin, & Shea, 2004) In addition,
substrate-mediated delivery can enhance gene transfer efficiency by
concentrating gene vehicles on biomaterials. (Hu, Lang, & Krebsbach,
2008) For example, intravascular stents have been polymer-coated to
direct gene delivery for treating vascular diseases. (Klugherz et al.,
2000) In another study, Hu et al. also showed that polyplex bound to
biomaterials using biotin-avidin interaction could significantly increase
transfection efficiency of non-viral vectors. (Hu, Syu et al., 2012)
Scaffolds are potential substrates for gene delivery. These bioma-
terial constructs are commonly applied in tissue engineering to provide
in vivo-mimic microenvironment. (Shrestha et al., 2016) If nonviral
vectors are immobilized onto their surfaces, adhered cells may directly
uptake genes to express biological cues and guide cell differentiation.
Therefore, in this study, we proposed to develop a multifunctional
scaffold which might not only provide an appropriate

⁎
Corresponding author at: Department of Chemical and Materials Engineering, National Central University, No. 300, Zhongda Rd., Zhongli District, Taoyuan City 32001, Taiwan.
E-mail address: huweiwen@cc.ncu.edu.tw (W.-W. Hu).

https://doi.org/10.1016/j.carbpol.2017.11.030
Received 19 July 2017; Received in revised form 5 October 2017; Accepted 8 November 2017
Available online 08 November 2017
0144-8617/ © 2017 Elsevier Ltd. All rights reserved.
W.-W. Hu et al.
microenvironment for cell growth but also mediate gene transfer.
Compared to sole biomaterial which is difficult to exhibit the char-
acteristics of both biocompatibility and gene delivery ability, composite
scaffolds should be more suitable to create an ideal environment for
these requirements because of their tunability. (Mukundan et al., 2015;
Tipduangta et al., 2015) Electrospinning is an attractive technique for
scaffold preparation due to its capability of fabricating various poly-
meric nanofibers. The prepared nanofibrous scaffolds not only exhibit
appropriate pore sizes and porosity to allow cell migrations but also
own sufficient surface area to encourage cell adhesion, proliferation
and differentiation. (Hu, Zuo, Zhang, Lan, & Zhang, 2012) Owing to the
feasibility of electrospinning for nanofibrous scaffold preparation, dif-
ferent polymer blends have been electrospun. (Mukundan et al., 2015;
Tipduangta et al., 2015) However, the compatibility between materials
restricts the blending ratio; therefore, only a limited composition can be
achieved. (Jeong et al., 2011) In order to overcome these difficulties,
some researchers have developed a dual-jet system to co-electrospin, by
which two different polymers can be deposited in arbitrary ratios by
adjusting their flow rates. The surface properties of materials can thus
be regulated according to the composition of scaffolds. (Hu & Yu, 2013)
Therefore, we proposed to develop a nanofibrous scaffold using
anionic alginate to adsorb cationic PEI/DNA nanocomplexes. However,
alginate is not an ideal material for cell growth due to its lack of cell
binding motif, and its negative charges also may inhibit cell adhesion.
(Lee & Mooney, 2012; Maroudas, 1975; Tallawi et al., 2015) To over-
come this drawback, the biocompatible polycaprolactone (PCL) was
incorporated using the coelectrospinning technique. (Poornima &
Korrapati, 2016) We hypothesized that genes immobilized on alginate
fibers might be uptaken by adhered cells for in situ transfection, and the
incorporated PCL might enhance cell viability. Additionally, the fabri-
cated composite nanofibrous scaffolds should be able to balance the
requirements of both gene delivery and biocompatibility, by adjusting
the ratios of alginate and PCL.
2. Material and methods
2.1. Materials
Alginic acid sodium salt from brown algae with medium viscosity
(viscosity > 2000 cps at 25 °C, molecular weight ranges between 80
and 120 kDa) was purchased from Sigma-Aldrich. The ratio of man-
nuronic acid to guluronic acid (M/G ratio) is 1.56. Polycaprolactone
(PCL) with molecular weight of 70–90 kDa, polyethylene oxide (PEO)
with molecular weight of 900 kDa, and 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), were also obtained from
Sigma-Aldrich. Fetal bovine serum (FBS), Dulbecco’s modified Eagle
medium (D-MEM), and trypsin were purchased from Gibco.
2.2. Preparation of alginate and PCL solutions
Stock solutions of sodium alginate and PEO were individually pre-
pared in distilled deionized (dd) water in concentrations of 6 and 10 wt.
%, respectively. Stock solutions of PCL was prepared in glacial acetic
acid in a concentration of 10 wt.%. These stock solutions were placed in
water bath at 60 °C for 1 day to accelerate their dissolution. They were
purified by a glass microfiber filter with a pore size of 0.7 μm (Grade
GF/F, Whatman). The co-solvent was prepared by dimethyl sulfoxide
(DMSO) and surfactant Triton X-100 at concentrations of 0.5 and 10 wt.
% in dd water, respectively. For 10 g of electrospun alginate solution
preparation, 6.7 g of 6 wt.% alginate stock solution, 2.5 g of 10 wt.%
PEO stock solution, and 0.8 g of co-solvent were mixed together to
make the final concentrations of 4 wt.% alginate and 2.5 wt.% PEO. On
the other hand, 10 g of electrospun PCL solution was prepared by
mixing 5 g of 10 wt.% PCL stock solution, 4 g of 10 wt.% PEO stock
solution, and 1 g of cosolvent so that the final concentrations were 5 wt.
% PCL and 4 wt.% PEO. These mixtures were thoroughly stirred for
30
Carbohydrate Polymers 183 (2018) 29–36
1 day; then, they were centrifuged to remove bubbles.
2.3. Electrospinning of nanofibers
The coelectrospining system was developed according to our pre-
vious study. (Hu & Yu, 2013) Electrospun alginate and PCL solutions
were fed into two 1 ml disposable syringes with 24G needle tips (inner
diameter = 0.29 mm) which were connected to two separate power
supplies (Chargemaster CH50-P, Simco) with identical voltages of
15 kV. Glass cover slips with diameters of 16 mm were placed on a
grounded stainless steel plate to collect electrospun fiber. The distances
between tips and the collector were 20 cm for all tests. To avoid the
interference between jets of alginate/PEO and PCL/PEO solutions, there
was a switch to control two power supplies and that each polymer so-
lution jetted for 15 s in turn. Pure alginate/PEO or PCL/PEO nanofibers
were prepared by single nozzle electrospinning, using the same condi-
tions as the dual jet system with the flow rate of 2 μl/min. The spun
nanofibers were dried in an oven at 37 °C for 1 day.
2.4. Immobilization of the nanoparticles onto nanofibrous scaffolds
Plasmid DNA encoding eGFP (pEGFP-C3, Clontech, CA, USA) and
PEI solutions were prepared in Tris-buffered saline (TBS) at con-
centrations of 80 and 104 μg/ml, respectively. Then, equal volumes of
DNA and PEI were mixed and vortexed thoroughly to prepare com-
plexed nanoparticles, so that the ratio of the amine groups from PEI to
the phosphate groups from DNA (N/P ratio) was 10. (Hu et al., 2013)
Before nanoparticle immobilization, nanofibers electrospun on cover
slips were immersed into absolute ethanol with 2% CaCl2 for 2 h to
crosslink alginate fibers. After TBS rinses, these fibers were placed in
24-well plates. Then, 500 μl of PEI/DNA nanoparticles were added per
well and incubated at 37 °C for 2 h. The unbounded nanoparticles were
removed through TBS rinses. For DNA labeling, Hoechst 33258 was
added to DNA at a concentration of 8 μg/ml before complexing with
PEI. The immobilized DNA were examined using fluorescent micro-
scopy (Eclipse Ti-U, Nikon, Japan), and the intensity of fluorescence
was quantified using a proper software (NIS Elements Basic Research,
Nikon, Japan).
2.5. Scanning electron microscopy (SEM)
Nanofibers were sputter-coated with gold and their morphologies
were observed by scanning electron microscopy (SEM, 3500N, Hitachi,
Japan). The diameters of nanofibers were evaluated by making 100
measurements based on their SEM images. In order to analyze the
collection rates at different feed rates, nanofibers were collected for
5 min, and the total lengths of the nanofibers in SEM images were
measured (NIS Elements Basic Research, Nikon, Japan), and then were
divided by the image area to determine the fiber density.
2.6. Fluorescence staining of nanofibers
To distinguish the distribution of different nanofibers on substrates,
fluorescein and rhodamin B were used to label alginate and PCL solu-
tions, respectively. These dyes were dissolved in dd water in con-
centrations of 1 wt.%, and 10 μl of stain solutions were added to 5 ml of
polymer solutions. The electrospinning was performed for 5 min, and
the collected nanofibers were observed using fluorescent microscopy.
2.7. Fourier transform infrared (FTIR) spectroscopy
Alginate, PCL, and composite nanofibers were electrospun for 2 h.
These fibrous mats were examined by Fourier transform infrared (FTIR)
spectroscopy (FT/IR 410, Jasco, USA) in a resolution of 4 cm−1 be-
tween 4000 and 600 cm−1.
W.-W. Hu et al.
2.8. Contact angle analysis
Water contact angles of electrospun nanofibers were determined by
Drop Shape Analysis system (DSA10, Kruss GmbH, Hamburg,
Germany). Four measurements per substrate were taken and data were
presented as averages with standard deviations.
2.9. Biocompatibility of nanofibers
Human embryonic kidney cell line (HEK-293T) (ATCC, Manassas,
VA, USA) was applied to evaluate biocompatibility of nanofibers. These
cells were seeded to electrospun scaffoldss placed in 24-well multiplates
at a density of 20,000 cells per well. They were cultured in DMEM
containing 10% FBS and were incubated at 37 °C with 5% CO2. Lactate
dehydrogenase (LDH) and MTT assays were performed to quantify the
cell numbers and cell viability, respectively.
The LDH assay for cell number quantification was determined by
CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega Madison,
WI, USA) according to the kit instructions for the total cell number
assay. Briefly, cell medium was removed and 75 μl of lysis buffer was
added to sample for 1 h at 37 °C to release LDH from live cells. Then,
50 μl of these lysates were moved to 96-well multiplates to react with
50 μl of LDH reagent for 30 min at room temperature. Finally, each well
was added 50 μl of stop solution and was analyzed spectro-
photometrically at 490 nm (Synergy H1, Biotek, USA). These results
were compared with a calibration curve made by lysing known cell
amounts.
For the MTT assay, 100 μl of MTT solution (5 mg/ml in PBS) was
added to the nanofiber samples with 900 μl of medium for 3 h at 37 °C.
Then, the supernatant was removed and 1 ml of DMSO was added to
dissolve formazan from cells, which was analyzed spectro-
photometrically at 550 nm.
2.10. In situ transfection experiments
In order to determine the efficiency of substrate-mediated gene
delivery, HEK-293T cells were seeded onto PEI/DNA adsorbed nanofi-
bers in a density of 20,000 cells per scaffolds. After culturing for 4 days,
the expression of eGFP from transfected cells was evaluated using
fluorescent microscopy, and the image software was applied to quantify
the intensity of fluorescence (NIS Elements Basic Research, Nikon,
Japan).
2.11. Statistical analysis
The statistical analyses were performed following a two-tailed
Student's t-test (SPSS, Chicago, IL, USA) to make comparison and the
errors were reported as standard deviations.
3. Results and discussion
3.1. The fabrication of PCL/alginate composite nanofibers in different ratios
In this study, a coelectrospinning technique was applied to prepare
multifunctional composite nanofibers for substrate-mediated gene de-
livery; biocompatible PCL was used to facilitate cell adhesion and an-
ionic alginate fibers allowed the adsorption of cationic PEI/DNA na-
noparticles (Fig. 1). The morphologies of electrospun PCL and alginate
were illustrated in Fig. 2. Compared to alginate fibers (289 ± 51 nm)
(Fig. 2a), diameters of PCL fibers (Fig. 2b) were thicker with broader
distribution (691 ± 116 nm). In addition, these fibers were placed in
TBS at 37 °C to examine their stability (Fig. S1). Their SEM results re-
vealed that although PEO was added to electrospun alginate and PCL
solutions, the prepared scaffolds maintained as fibers in aqueous en-
vironment. Then, different perfusion rates of polymer jets were ex-
amined to determine their effects on fiber deposition rates (Fig. 3a). The
31
Carbohydrate Polymers 183 (2018) 29–36
results demonstrated that the fiber collection rates linearly depended on
the perfusion rates. In addition, the slope of the regression line of the
alginate groups was 8 times that of the PCL groups. These results sug-
gested that the deposited alginate fibers would be 8 times as many as
the PCL fibers when they were electrospun in the same perfusion rate.
Therefore, we applied three different perfusion rates to coelectrospin
composite nanofibers in compositions of 80% alginate/20% PCL, 50%
alginate/50% PCL, and 20% alginate/80% PCL, which were denoted as
A8P2, A5P5, and A2P8, respectively (Table 1). The SEM images showed
that nanofibers with higher ratios of PCL demonstrated more thick fi-
bers (Fig. 3b) which were consistent with the characteristics of PCL
fibers (Fig. 2b). Further fluorescence staining was also applied to
identify deposited nanofibers; hence, alginate and PCL solutions for
coelectrospinning were added green and red fluorescent dyes, respec-
tively. The fluorescent images demonstrated that alginate and PCL fi-
bers were evenly distributed on the collector, and the respective com-
position was consistent with the target ratios because the groups with
higher ratios of alginate fibers showed more red lines in the images
(Fig. 3c). These results suggested that through the control of perfusion
rates of polymer jets during coelectrospinning, composite nanofibers in
defined ratios can be easily prepared.
3.2. Material properties of composite nanofibers
Since coelectrospinning can prepare composite fibers in specific
ratios, it should be interesting to investigate their properties. Therefore,
FT-IR analysis was applied, and the spectra of PCL and alginate fibers
both demonstrated their characteristic absorptions (Fig. 4a). The spe-
cific peaks of PCL were appeared at 2950, 2860, and 730 cm−1, re-
vealing the asymmetric stretching, symmetric stretching, and long
chain rocking motion of vibrations of CH2, respectively. (Elzein, Nasser-
Eddine, Delaite, Bistac, & Dumas, 2004; Peng, Han, Liu, Tjiu, & He,
2010) In addition, its stretching of carbonyl group appeared at
1725 cm−1. (Elzein et al., 2004) Regarding alginate fibers, a board peak
appeared at 3400 cm−1 due to the stretching vibration of eOH groups.
Further, the symmetric and asymmetric stretching of carboxyl groups of
alginate exhibited peaks at 1610 and 1415 cm−1, respectively.
(Sarmento, Ferreira, Veiga, & Ribeiro, 2006) In what concerns the
composite fibers, they exhibited specific peaks of both PCL and algi-
nate, and these peaks were directly proportional to the composition
ratios. These results suggested that coelectrospinning not only evenly
distributed PCL and alginate on substrate but also modulated the che-
mical properties of composite nanofibers according to the composition
ratios.
In addition to FT-IR, water contact angle analysis was also per-
formed in order to determine hydrophobicity of nanofibers (Fig. 4b). It
could be verified that alginate fibers were extremely hydrophilic as they
presented an extremely small water contact angle (19.5 ± 0.7°). In
contrast, the large water contact angle of PCL fibers (72.2 ± 1.4°)
suggested its hydrophobic property. In what concerns the composite
nanofibers, it could be observed that their hydrophobicity depended on
the respective compositions: the higher PCL ratios exhibited the greater
water contact angles. These results suggested that the surface properties
of composite fibers could be easily controlled by the fiber ratios.
3.3. The DNA adsorption on composite nanofibers
Because carboxyl groups of alginate exhibited negative charges,
cationic PEI/DNA nanoparticles should be capable of being adsorbed
onto those anionic fibers. To examine this hypothesis, PEI/DNA nano-
particles were added to alginate, PCL, or composite nanofibers. These
DNA molecules were stained by Hoechst 33258 before complexing with
PEI to determine the immobilization efficiency of gene carriers. After
2 h incubation, these fibers were thoroughly washed by TBS to remove
unbound nanoparticles, and the adsorbed nanoparticles appeared as
blue spots under a fluorescent microscope (Fig. 5a). The quantification
W.-W. Hu et al. Carbohydrate Polymers 183 (2018) 29–36

Fig. 1. The scheme of multifunctional composite nanofibers

for in situ transfection.

of fluorescent images was also evaluated by software for comparison. more DNA. These results suggested that positively charged PEI/DNA
There was no blue spot on pure PCL fibers. In contrast, the pure alginate nanocomplexes were specifically adsorbed onto alginate fibers.
fibers demonstrated the highest immobilized DNA result. For the In addition, the adsorption of PEI/DNA nanoparicles onto composite
composite fibers, the groups with higher ratios of alginate maintained fibers was illustrated by SEM (Fig. 5b). Nanoparticles ranged from 400

Fig. 2. The morphologies and size distributions of electrospun (a) alginate and (b) PCL nanofibers.

32
W.-W. Hu et al. Carbohydrate Polymers 183 (2018) 29–36

Fig. 3. Coelectrospun nanofibers. (a) Alginate and PCL nanofibers were electrospun individually on glass cover slips for 5 min. The deposition rates of nanofibers were linearly correlative
to the perfusion rate of polymer jets during coelectrospinning. (b) Composite nanofibers in different ratios were prepared by a dual-jet coelectrospinning system, and their morphologies
were illustrated by SEM. The groups with higher ratios of PCL showed more thick fibers. (c) Polymer solutions were stained by fluorescent dyes before coelectrospinning (green: alginate;
red: PCL). The groups with higher ratios of alginate fibers showed more red lines in the images. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

Table 1 3.4. Biocompatibility of composite nanofibers

The perfusion rates of polymer jets for regulating the deposition rates of alginate and PCL
fibers.
Although we have demonstrated that alginate fibers could im-
Alginate PCL mobilize cationic non-viral vectors, its hydrophilic properties may in-
hibit protein adsorption and thus reduce cell adhesion. Therefore, PCL
Perfusion rate Deposition rate Perfusion rate Deposition rate was incorporated to nanofibers to improve biocompatibility. In order to
(μl/min) (μm/μm2–h) (μl/min) (μm/μm2 −h) evaluate the effect of PCL on cell adhesion, HEK293T cells were seeded
A8P2 1.00 2.46 ± 0.12 2.0 0.63 ± 0.18 on nanofibers for 3 days, and cell images were captured using phase
A5P5 0.50 1.26 ± 0.10 4.0 1.25 ± 0.12 contrast microscopy and SEM. The phase contrast microscopy images
A2P8 0.25 0.63 ± 0.06 8.0 2.46 ± 0.18 illustrated that cells on alginate fibers were aggregated as spheroids
with low level of cell spreading (Fig. 6a). In contrast, PCL seemed to be
relatively biocompatible because cells grown on PCL fibers demon-
to 600 nm diameters were found on composite nanofibers. Further- strated epithelial-like morphology. For the composite fibers, cell ex-
more, these nanoparticles were only adsorbed on thin nanofibers which tension was determined by the fiber composition as cell morphologies
were consistent to the size of alginate fiber we reported in Fig. 2a. changed from spheroid-form to epithelial-form when the ratios of PCL
fibers were increased. The SEM results also suggest this trend (Fig. 6b).
In contrast to the alginate fiber group, cells grown on PCL nanofibers

Fig. 4. Characteristics of coelectrospun nanofibers. (a) Chemical functional groups of electrospun nanofibers were examined by FTIR analysis. The specific peaks of alginate and PCL were
indicated using black dashed lines and red dotted lines, respectively. (b) Water contact angle were applied to determine the wettability of nanofibers. The higher ratios of PCL led to the
larger water contact angles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

33
W.-W. Hu et al. Carbohydrate Polymers 183 (2018) 29–36

Fig. 5. Gene immobilization onto composite nanofibers. (a) To illustrate gene immobilization, DNA was stained by Hoechst 33258 before complexing with PEI. Cationic PEI/DNA
nanoparticles were adsorbed onto composite nanofibers and unbound ones were remove by thoroughly TBS washes. Fluorescent images showed that fibers with higher ratios of alginate
maintained more blue spots. Their quantification results were also provided (**: p < 0.01) (b) The adsorption of nonviral vectors on composite nanofibers were examined by SEM. These
images showed that nanoparticles ranged from 400 to 600 nm diameters were immobilized only on thin fibers. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)

exhibited abundant pseudopodia to adhere onto nanofibers. Therefore,
the more PCL in composite fibers resulted in the higher levels of cell
extension.
In addition to cell morphology, LDH and MTT assays were also
applied to determine cell numbers and cell activity, respectively
(Fig. 6c). According to the LDH assay, cells grown on PCL fibers were
more in number than those from alginate fibers. The amount of cells on
composite nanofibers slightly increased with PCL ratios although the
differences between them were not significant. Additionally, the MTT
results also indicated that PCL fibers highly increased cell activity
compared to alginate fibers. Interestingly, MTT results demonstrated
that the activity of cells on composite fibers increased with the PCL
ratios. According to the cell morphology results (Fig. 6a,b), the im-
provement of cell activity by PCL component likely corresponded to its
promotion on cell extension. Therefore, the incorporation of PCL should
be beneficial to enhance scaffold biocompatibility.
3.5. The application of composite nanofibers for in situ transfection
According to previous experiments, composite fibers demonstrated
their gene immobilization ability (Fig. 5) and their biocompatibilities
can be manipulated through the PCL component (Fig. 6). Therefore,
these scaffolds were applied in this study for in situ transfection. Before
cell seeding, PEI/DNA nanoparticles were adsorbed onto alginate, PCL,

Fig. 6. The effects of nanofiber composition on cell morphology and biocompatibility. Different nanofibers were used to culture HEK-293T cells, and the morphologies of surface cell were
illustrated by (a) phase contrast microscopy and (b) SEM. The higher ratios of PCL in composite fibers demonstrated the better cell adhesion and extension. (c) Cells cultured on fibers
were examined by LDH and MTT to determine cell number and cell activity, respectively. Both results increased with the PCL ratios, suggesting the improvement of PCL on bio-
compatibility of scaffolds. (t tests compared to the results of alginate fibers. #: p < 0.05 for the LDH results; *: p < 0.05, **: p < 0.01 for the MTT results;).

34
W.-W. Hu et al.
or composite nanofibers, and the unbound ones were then thoroughly
washed by TBS. Afterward, HEK293T cells were cultured on these gene
immobilized scaffolds for 3 days to allow in situ transfection. The
transfected cells on these scaffolds were observed using fluorescent
microscopy (Fig. 7a). In contrast to anionic alginate fibers, neutral PCL
fibers were incapable of adsorbing PEI/DNA nanoparticles, and thus
there was no transfected cell found on PCL fibers. Regarding the com-
posite fibers, it can be verified that the levels of trangene expression
depended on the alginate component that the higher ratios of alginate
resulted in the more transfected cells. The fluorescence of these images
was quantified, and the results suggested that GFP expressed cells on
composite fibers were directly proportional to the alginate ratios
(Fig. 7b). Among these composite fibers, A8P2 demonstrated the best
transgene expression, which almost the same as that of the pure algi-
nate group (Fig. 7b). In addition, bioactivity of A8P2 was also im-
proved, both in cell adhesion and cell extension (Fig. 7a). These results
suggested that the biocompatibility of alginate scaffolds may be im-
proved by incorporating with slight PCL fibers without hindering their
in situ transfection ability.
4. Conclusions
In this study, it was verified that alginate and PCL can be evenly
spreaded to form composite nanofibrous scaffolds, and that by reg-
ulating the perfusion rates of polymer jets, alginate/PCL fibers can be
prepared in specific ratios. Therefore, their chemical and surface
properties may be controlled by manipulating the composition. Because
anionic alginate can adsorb cationic PEI/DNA nanoparticles, these
composite fibers demonstrated their potential to be applied for in situ
transfection. Further, the incorporation of PCL in the composites can
improve cell adhesion and extension to enhance biocompatibility.
Hence, by optimizing the ratios of PCL and alginate, multifunctional
scaffolds may thus be prepared to balance the requirements of gene
delivery and biocompatibility. The composite scaffold system
35
Carbohydrate Polymers 183 (2018) 29–36
Fig. 7. In situ transfection on nanofibers. (a) After
immobilizing DNA/PEI nanoparticles, HEK-293T
cells were seeded on nanofibers and cultured for
3 days, and the GFP expression from tranfected cells
was evaluated using fluorescent microscopy. The
results suggested the transfection efficiency of com-
posites fibers increased with their alginate ratios. (b)
The quantification of GFP from fluorescent images
also showed the same trend. (**: p < 0.01). (For
interpretation of the references to colour in this
figure legend, the reader is referred to the web ver-
sion of this article.)
developed in this study can be easily prepared and broadly used for
regenerative gene therapy. Furthermore, its tunability is beneficial to
custom-tailoring according to different clinical requirements, which
may be highly potential for tissue engineering applications.
Acknowledgements
This work was supported by the grant of MOST 105-2221-E-008-
110- from the Ministry of Science and Technology of Taiwan and 105
CGH-NCU-A2 from the National Central University and Cathy General
Hospital Joint Research Center.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.carbpol.2017.11.030.
References
Chertok, B., Langer, R., & Anderson, D. G. (2016). Spatial control of gene expression by
nanocarriers using heparin masking and ultrasound-targeted microbubble destruc-
tion. ACS Nano, 10(8), 7267–7278.
Crystal, R. G. (1995). Transfer of genes to humans − Early lessons and obstacles to
success. Science, 270(5235), 404–410.
Elzein, T., Nasser-Eddine, M., Delaite, C., Bistac, S., & Dumas, P. (2004). FTIR study of
polycaprolactone chain organization at interfaces. Journal of Colloid and Interface
Science, 273(2), 381–387.
Hu, W. W., & Yu, H. N. (2013). Coelectrospinning of chitosan/alginate fibers by dual-jet
system for modulating material surfaces. Carbohydrate Polymers, 95(2), 716–727.
Hu, W. W., Wang, Z., Hollister, S. J., & Krebsbach, P. H. (2007). Localized viral vector
delivery to enhance in situ regenerative gene therapy. Gene Therapy, 14(11),
891–901.
Hu, W. W., Lang, M. W., & Krebsbach, P. H. (2008). Development of adenovirus im-
mobilization strategies for in situ gene therapy. Journal of Gene Medicine, 10(10),
1102–1112.
Hu, W. W., Ward, B. B., Wang, Z., & Krebsbach, P. H. (2010). Bone regeneration in defects
compromised by radiotherapy. Journal of Dental Research, 89(1), 77–81.
Hu, W. W., Lin, Z. W., Ruaan, R. C., Chen, W. Y., Jin, S. L. C., & Chang, Y. (2013). A novel
application of indolicidin for gene delivery. International Journal of Pharmaceutics,
W.-W. Hu et al.
456(2), 293–300.
Hu, W. W., Wang, Z., & Krebsbach, P. H. (2016). Virus immobilization on biomaterial
scaffolds through biotin-avidin interaction for improving bone regeneration. Journal
of Tissue Engineering and Regenerative Medicine, 10(2), E63–E72.
Hu, W. W., Syu, W. J., Chen, W. Y., Ruaan, R. C., Cheng, Y. C., Chien, C. C., et al. (2012).
Use of biotinylated chitosan for substrate-mediated gene delivery. Bioconjugate
Chemistry, 23(8), 1587–1599.
Hu, A., Zuo, B., Zhang, F., Lan, Q., & Zhang, H. (2012). Electrospun silk fibroin nanofibers
promote Schwann cell adhesion, growth and proliferation. Neural Regeneration
Research, 7(15), 1171–1178.
Huang, Y. C., Simmons, C., Kaigler, D., Rice, K. G., & Mooney, D. J. (2005). Bone re-
generation in a rat cranial defect with delivery of PEI-condensed plasmid DNA en-
coding for bone morphogenetic protein-4 (BMP-4). Gene Therapy, 12(5), 418–426.
Jang, J. H., Houchin, T. L., & Shea, L. D. (2004). Gene delivery from polymer scaffolds for
tissue engineering. Expert Review of Medical Devices, 1(1), 127–138.
Jeong, S. I., Krebs, M. D., Bonino, C. A., Samorezov, J. E., Khan, S. A., & Alsberg, E.
(2011). Electrospun chitosan-alginate nanofibers with In situ polyelectrolyte com-
plexation for use as tissue engineering scaffolds. Tissue Engineering Part A, 17(1–2),
59–70.
Klugherz, B. D., Jones, P. L., Cui, X. M., Chen, W. L., Meneveau, N. F., DeFelice, S., et al.
(2000). Gene delivery from a DNA controlled-release stent in porcine coronary ar-
teries. Nature Biotechnology, 18(11), 1181–1184.
Lee, K. Y., & Mooney, D. J. (2012). Alginate: Properties and biomedical applications.
Progress in Polymer Science, 37(1), 106–126.
Lee, K., Silva, E. A., & Mooney, D. J. (2011). Growth factor delivery-based tissue en-
gineering: General approaches and a review of recent developments. Journal of the
Royal Society Interface, 8(55), 153–170.
Maroudas, N. G. (1975). Adhesion and spreading of cells on charged surfaces. Journal of
Theoretical Biology, 49(2), 417–424.
36
Carbohydrate Polymers 183 (2018) 29–36
Mukundan, S., Sant, V., Goenka, S., Franks, J., Rohan, L. C., & Sant, S. (2015).
Nanofibrous composite scaffolds of poly(ester amides) with tunable physicochemical
and degradation properties. European Polymer Journal, 68, 21–35.
Munoz Ruiz, M., & Regueiro, J. R. (2012). New tools in regenerative medicine: Gene
therapy. Advances in Experimental Medicine and Biology, 741, 254–275.
Peng, H., Han, Y., Liu, T., Tjiu, W. C., & He, C. (2010). Morphology and thermal de-
gradation behavior of highly exfoliated CoAl-layered double hydroxide/poly-
caprolactone nanocomposites prepared by simple solution intercalation.
Thermochimica Acta, 502(1–2), 1–7.
Poornima, B., & Korrapati, P. S. (2016). Fabrication of chitosan-polycaprolactone com-
posite nanofibrous scaffold for simultaneous delivery of ferulic acid and resveratrol.
Carbohydrate Polymers, 157, 1741–1749.
Sarmento, B., Ferreira, D., Veiga, F., & Ribeiro, A. (2006). Characterization of insulin-
loaded alginate nanoparticles produced by ionotropic pre-gelation through DSC and
FTIR studies. Carbohydrate Polymers, 66(1), 1–7.
Shrestha, B. K., Mousa, H. M., Tiwari, A. P., Ko, S. W., Park, C. H., & Kim, C. S. (2016).
Development of polyamide-6,6/chitosan electrospun hybrid nanofibrous scaffolds for
tissue engineering application. Carbohydrate Polymers, 148, 107–114.
Tallawi, M., Rosellini, E., Barbani, N., Cascone, M. G., Rai, R., Saint-Pierre, G., et al.
(2015). Strategies for the chemical and biological functionalization of scaffolds for
cardiac tissue engineering: A review. Journal of the Royal Society Interface, 12(108),
21050254.
Tipduangta, P., Belton, P., Fabian, L., Wang, L. Y., Tang, H., Eddleston, M., et al. (2015).
Electrospun polymer blend nanofibers for tunable drug delivery: The role of trans-
formative phase separation on controlling the release rate. Molecular Pharmaceutics,
13(1), 25–39.
Vo, T. N., Kasper, F. K., & Mikos, A. G. (2012). Strategies for controlled delivery of growth
factors and cells for bone regeneration. Advanced Drug Delivery Reviews, 64(12),
1292–1309.