A Controllable Local Drug Delivery System Based On Porous Fibers For Synergistic Treatment of Melanoma and Promoting Wound Healing

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Page 1 of 25 Biomaterials Science
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A Controllable Local Drug Delivery System Based on Porous Fibers DOI: 10.1039/C9BM01045A
for Synergistic Treatment of Melanoma and Promoting Wound
Healing
Biomaterials Science Accepted Manuscript

Zhipeng Yuana, Kexin Zhanga, Xiangyu Jiaoa, Yaru Chenga, Yiyi Zhanga, Peixun
Zhangb, Xueji Zhanga, Yongqiang Wena,*
a. Research Center for Bioengineering & Sensing Technology, School of Chemistry and Biological
Engineering, University of Science and Technology Beijing, Beijing 100083, China
b. Department of Orthopedics and Trauma, Peking University People’s Hospital, Beijing 100083,
Published on 14 September 2019. Downloaded on 9/17/2019 1:54:18 AM.
China
* for corresponding author.
E-mail address: wyq_wen@ustb.edu.cn
Abstract
A dual function system that inhibits tumor growth while promoting wound healing is
very necessary for melanoma treatment since tumor killing and skin healing are two
complementary and influential processes. Herein, a controllable local drug delivery
system based on porous fiber membrane incorporated with CuS nanoparticles is
designed for chemo-photothermal synergistic melanoma therapy and promoting wound
healing. The porous structure on fiber surface significantly increases the drug loading
capacity of the membrane and the photothermal effect of incorporated CuS
nanoparticles is used to control the drug release rate. Benefit from the chemo-
photothermal synergistic therapy, the composite membrane can effectively kill
melanoma cells in vitro and inhibit tumor growth in vivo. Furthermore, the membrane
can also significantly promote the cutaneous wound healing by providing mechanical
support and releasing of copper ions. Thus, this work provides new ideas for the
development of multifunctional local treatment and postoperative care systems.
Keywords: porous fiber; controlled drug delivery; synergistic melanoma therapy;
wound healing
1. Introduction
Melanoma is a lethal skin cancer and notorious for its aggressive, highly metastatic
and treatment-resistant features [1]. Surgical excision is the most commonly used and
preferred treatment options for melanoma patients. However, local recurrence arising
from the nearby covert tumor cells after surgery remains a great challenge [2-4].
Therefore, the other therapies such as chemotherapy [5], photothermal therapy [6],
immunotherapy [7], gene therapy [8, 9] and their synergistic therapy [10, 11] were also
common options to treat melanoma or as adjuvant therapies after surgery to reduce the
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Biomaterials Science Page 2 of 25
risk of recurrence. Since the surgery resection could inevitably cause cutaneousDOI:
defects,View Article Online
10.1039/C9BM01045A
immediate repair of damaged tissue is of great importance for long-term healing.
Therefore, a multi-function system for simultaneously treating melanoma and
promoting wound healing is in urgent necessary.
The systemic therapy systems based on particulate form for internal tumors had been
extensively explored. However, some disadvantages like low target efficiency to the
tumor site and detrimental side effects limit its clinical application. For melanoma, local

therapy is a more suitable method since it grows on superficial skin. The local therapy
system can minimize side effects to normal tissues by avoiding excessive circulation
[12]. Recently, various anticancer systems for local melanoma therapy have been
widely studied [13-16]. Among them, electrospun scaffolds are gradually favored due
to its specific properties such as high specific surface area, high porosity, diverse
composition, controllable and adjustable microstructures [17, 18]. Electrospun fibers
are commonly used as drug delivery systems for chemotherapy. The incorporation of
photothermal reagents [19, 20] into the fibers can impart photothermal properties to the
fiber membrane, thereby achieving the purpose of chemo-photothermal synergistic
treatment to improve the therapeutic effect. The combination of PTT and chemotherapy
can accelerate tumor cell inactivation as the high temperature increases the permeability
of cell membrane, thus promote absorption of drugs [12]. Moreover, in recent
researches, electrospinning scaffolds also showed great potential in wound healing as
it can mimic hierarchical architecture and properties of the native tissue extracellular
matrice [21, 22]. The scaffolds can provide both mechanical support and structural cues
for cell adhesion, migration and proliferation [23, 24].
The drug-loaded electrospun fibers are commonly prepared by co-spinning drugs
with polymer or constructing a core-shell structure [25]. They rely mainly on the
degradation of the fiber to release the drug, so the release behavior extends throughout
almost the entire degradation period of the scaffold. Some studies have loaded drugs
onto the fiber surface by physical adsorption or chemical immobilization [26]. But this
is difficult to ensure the drug stability and achieve controlled release of the drug. Due
to all these fibers have a smooth surface, drug loading capacity is also limited. For the
treatment of melanoma, tumor killing and skin healing are complementary. Treating the
tumor while healing the skin is the goal we pursue. Therefore, a suitable amount of
chemotherapy drugs is important. When the drug is insufficient, the tumor killing
efficiency is low and it is difficult to inhibit tumor growth. However, when the drug is
excessive, the chemotherapeutic drug is released for a long time, and since the
chemotherapy drug has certain cytotoxicity, it is unfavorable for skin healing. Thus, it
is important to further improve the regulation of drug loading and the controllability of
drug release.
In this article, porous fiber membrane incorporated with CuS nanoparticles was
prepared for chemo-photothermal synergistic melanoma therapy and wound healing.
Porous fibers were first prepared by electrospinning method combined with breath
figure mechanism. DOX as chemotherapeutic drug was loaded into the pores on the
fiber surface. The fibers were coated with a layer of PVA polymer after drug loading.
In this system, since the fiber surface has a porous structure, the drug-loading capacity
2
is enhanced and the drug loading can be adjusted as needed. Since the CuSView Article Online
DOI: 10.1039/C9BM01045A
nanoparticles are wrapped in the fibers, the monolithic nature of the membrane can ease
the retention at the tumor site and prevent the migration of nanoparticles. Thus the NIR
induced hyperthermia can repeatedly and effectively treat tumors for a long time. The
photothermal effect of incorporated CuS nanoparticles can be used to control the drug
release rate. Compared with coaxial fibers or co-spun fibers, the as-prepared fibers are
only coated with a thin layer of PVA, and the drug release behavior is easier to control.

Thus the composite porous fiber membrane overcomes the shortcoming of uncontrolled
slow drug release in common fiber drug delivery system. Benefit from the chemo-
photothermal synergistic therapy, the composite membrane showed robust anticancer
effects both in vivo and in vitro. At the same time, the fiber membrane could effectively
accelerate the wound healing in tumor-bearing and full-thickness skin defect mice. The
layer of PVA on fiber surface increased the hydrophilicity of the fiber membrane and
made it fit well to the skin. The fibers can not only provide mechanical support and
structural cues for cell proliferation but also continuously release therapeutic copper
ions [27] for wound healing. This composite porous fiber membrane provides a new
paradigm for controllable local drug delivery system and has great potential for
comprehensive treatment of melanoma and postoperative care after melanoma surgical
resection.
2. Experimental Section
2.1. Materials
PLA (Mw=3.5×105) and PCL (Mw=3.5×105) were purchased from Jinan Daigang
Co. Ltd. (Shandong, China). Dichloromethane (DCM), N, N-dimethylformamide
(DMF), were purchased from Innochem Technology Co. Ltd. (Beijing, China). Sodium
citrate dehydrate (C6H5Na3O7·2H2O), cupric chloride dihydrate (CuCl2·2H2O), sodium
sulfide nonahydrate (Na2S·9H2O), doxorubicin hydrochloride (≥98%) and polyvinyl
alcohol (PVA) were all purchased from Aladdin Bio-Chem Technology Co. Ltd.
(Shanghai, China). All buffers were prepared with ultra-pure Milli-Q water
(resistance>18 MΩ cm-1). All reagents were used as received.
2.2. Preparation and Conditional Exploration of Porous PLA/PCL Fiber Membrane

PLA/PCL porous fiber membranes were prepared by electrospinning method.
PLA/PCL (w:w=3:1, 1:1, 1:3) were respectively dissolved in the mixture solvent of
DCM/DMF(98:2), and the total polymer concentrations were all 8 wt%. After stirred
for 2 h at room temperature, the spinning solutions with various polymer ratio were
obtained. Then, PLA/PCL(w:w=3:1) were respectively dissolved in mixture solvent of
DCM /DMF with different ratios (v:v= 98:2, 95:5, 90:10). The total polymer
concentrations of the solutions were also 8 wt%. After stirred for 2 h at room
temperature, the spinning solutions with various solvent ratio were obtained. The
solutions were respectively loaded into a 20 ml syringe equipped with a stainless-steel
needle and discharged by a digitally controlled syringe pump, the flow rate and applied
3
voltage were set at 2 mL h-1 and 15 kV. The fibers were collected by aluminum foil.View Article Online
DOI: 10.1039/C9BM01045A
The experiment was carried out at 25°C and the environment relative humidity was
adjusted to 20%, 50%, 80% by humidifier and dehumidifier.
2.3. Synthesis of CuS NPs
The CuS nanoparticles were synthesized through previously reported method and

make some modifications [28]. In 1000 mL CuCl2·2H2O (10 mM) and sodium citrate
(6.8 mM) aqueous solution, 10 mL sodium sulfide solution (Na2S, 1 M) was added with
stirring at room temperature, and the CuCl2 solution turned dark-brown immediately.
Continue stirring for 10 minutes, then the mixture was heated to 90°C and stirred for
another 30 min, a dark green solution with precipitate was obtained. The mixture was
transferred to ice-cold water. After centrifugation and washing with ultrapure water for
three times, the black precipitate was lyophilized, and the CuS nanoparticles were
obtained.
2.4. Preparation of CuS Incorporated PLA/PCL PNMs
Various amounts of CuS nanoparticles (mass fraction of 0%, 15%, 30%, 45%
compared to polymer) were pre-mixed with the electrospinning solution and stirred for
2 h. The CuS nanoparticles incorporated PLA/PCL porous fiber membranes were
prepared by electrospinning with the optimum parameters for pore-forming obtained
above.
2.5. Drugs Loading and NIR-Triggered Release in Vitro
SNM, PNM and 30PNM were respectively immersed in 10 ml doxorubicin

hydrochloride solution (0.25 mg ml-1) with mixed solvent of water and ethanol (1:1)
and ultrasonic oscillation for 0.5 h. Then, the drug loaded membranes were taken out
and washed with water for three times to remove free drugs. After lyophilized, the drug
loaded membranes were immersed in 3% PVA aqueous solution and ultrasonic
oscillation till the membrane completely wetted. Finally, the membranes were dried in
a vacuum oven at 50°C.
To determine the drug-loading capacity of SNM, PNM and 30PNM, the supernatants
and washing solutions were collected. The loading capacity was quantified: load
capacity (mg/g) = (total DOX-unloaded DOX)/total weight of the membrane. DOX
concentrations were detected by fluorescence spectrophotometer (F-7000, Hitachi,
Japan).
For the measurement of in vitro NIR-triggered drug release, 30PNMP-D and PNMP-
D were immersed in PBS buffers and irradiated with NIR laser at power densities of
0.5 W cm-2 for 30min, then turn off the laser and maintained for 1 h. The operation
process was repeated for 4 cycles, at determined time intervals, the solution was
collected for fluorescence detection and replaced with fresh PBS buffers. The
percentage accumulative released DOX was quantified: accumulative release (%) =
4
(accumulative release DOX amount/total DOX amount) × 100%. View Article Online
DOI: 10.1039/C9BM01045A
2.6. Material Characterization
The morphologies and elemental distribution were observed by scanning electron

microscope (SEM, HITACHI, SU-8010, Japan) equipped with an energy-dispersive
spectrometer (EDS, IXRF SDD3030) and transmission electron microscope (TEM,

HITACHI, HT7700 Exalens, Japan). The crystal structure was characterized by X-ray
diffraction (XRD, BRUKER, D8 ADVANCE, Germany). The wettability of the
membranes was characterized by contact angle system (Data physics, OCA ES r,
Germany). Functional groups of membranes were tested by fourier transform infrared
spectrometer (Thermo Scientific, Nicolet 6700, U.S.A). To study the Cu ion release
from the membrane, PFMP, 30PFMP and 30PFMP-D were respectively immersed in 1
mL PBS and kept at 37 °C for 4 days. The supernatants were collected and the
concentration of Cu ions was measured by inductively coupled plasma-optical emission
spectrometry (ICP-OES, Agilent 5110, U.S.A) every day.
2.7. Photothermal Performance of PNMPs
The photothermal performance of PFMP, 15PFMP, 30PFMP, 45PFMP which

containing different amounts of CuS nanoparticles was measured in dry and wet (1ml
PBS) conditions respectively. The membranes were cut into circular shapes (diameter
15 mm), then placed in 24-well plate and exposed to 980nm laser (0.5 W·cm-2) for 2min
(dry) and 10min (wet). The temperature changes were monitored by digital
thermometer. The thermal images were recorded by a thermal infrared imager (Fluke,
Ti480, USA) in real time. The temperature changes of 30PFMP irradiated with various
laser power densities (0.05, 0.1, 0.3, 0.5 and 0.7 W cm-2) under wet conditions were
also tested. The photostability of 30PFMP was verified by irradiating the membrane
over four cycles under wet condition.
2.8. In Vitro Anticancer Effect
Mouse B16F10 melanoma cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM, Hyclone, USA) supplemented with 10% (v/v) fetal bovine serum
(FBS). Cells were maintained in a 5% carbon dioxide incubator at 37°C. 30PFMP,
30PFMP-D, 30SFMP-D were cut into circular shapes suitable for the pore size of 24-
well plate and sterilized for 30 min with UV light. Then six groups were set: control,
30PFMP, 30PFMP-D, 30PFMP+Laser, 30SFMP-D+Laser, 30PFMP-D+Laser. 5×104
cells were seeded in 24-well plates per well for 24 h, then corresponding membrane for
each group was added to the wells and the groups 30PFMP+Laser, 30SFMP-D+Laser,
30PFMP-D+Laser were irradiated with the 980nm NIR at the power density of 0.50 W
cm-2 for 15 min. Then the cells of all groups were cultured under the same conditions.
After 24h, cell viability was measured using the CCK-8 assay. The live/dead assay
(Calcein-AM/PI Double Stain Kit, Yeasen, China) were carried out to further confirm
5
the CCK-8 results. The cell suspension was mixed with the appropriate amount ofView Article Online
DOI: 10.1039/C9BM01045A
staining solution and incubated at 37°C for 15 min according to the manufacturer’s
instruction and observed with laser scanning confocal microscope (OLYMPUS
FV1000, Japan).
2.9. In Vivo Antitumor Efficacy and Wound Healing Evaluation

The BALB/c nude mice (female, 6 weeks) were purchased from Beijing Vital River
Laboratory Animal Technology Co., Ltd. and all animal procedures were performed in
accordance with the Guidelines for Care and Use of Laboratory Animals of Peking
University Health Science Center and approved by the Animal Ethics Committee of
Peking University Health Science Center. To establish the melanoma tumor model,
2×106 B16F10 cells in 100 μL of PBS were subcutaneously injected to the hind part of
each mouse. When the tumor sizes reached about 80 mm3, the mice were randomly
divided into six groups (n=5 per group): control, 30PFMP, 30PFMP-D, 30PFMP+Laser,
30SFMP-D+Laser, 30PFMP-D+Laser. A circular full-thickness skin wound with
diameter 10 mm was created at the tumor site. The wound area was covered with
corresponding membrane according to the grouping. 30PFMP+Laser, 30SFMP-
D+Laser and 30PFMP-D+Laser groups were irradiated with a 980 nm NIR laser (0.5
W cm-2, 10 min) once a day for consecutive 3 days (day 0, day 1, day 2). The tumor
surface temperature was recorded by a thermal infrared imager. The tumors and wounds
were photographed every 4 days. The length and width of the tumors were measured
by a vernier caliperevery 2 days, the tumor volume was calculated by: tumor volume =
(tumor length) × (tumor width)2/2. The mouse body weights were measured every 2
days. On day 14, the mice were sacrificed, tumor tissues were harvested, weighed, and
photographed. Then the tumor tissues were fixed in 4% paraformaldehyde solution,
gradient ethanol dehydrated, embedded in paraffin, and sectioned into 5 μm thick slices.
Histological analysis of tumor tissues was evaluated by H&E staining. Ki67 IHC
staining was used to evaluated the tumor cell proliferation. The slides were evaluated
using an optical microscope. A TUNEL assay was performed to observe cell apoptosis.
the TUNEL-stained slides were visualized on CLSM.
2.10 In Vitro Cell Adhesion and Proliferation
Mouse 3T3 fibroblasts and JB6 epidermal cells (ATCC) were cultured in DMEM
supplemented with 10% FBS. Cells were maintained in a 5% carbon dioxide incubator
at 37°C. To evaluate the cell adhesion, the fiber membranes were cut into circular shape
suitable for the pore size of 24-well plate and sterilized for 30 min with UV light. Then,
2 × 104 3T3 and JB6 cells per well were seeded onto the 30PFMP, 30PFMP-D after
DD and cultured for 24 h. It should be explained that "30PFMP-D after DD" refers to
30PFMP-D that has undergone a drug delivery period. During the drug delivery period,
the membrane was irradiated by 980nm laser (0.5 W cm-2) for 15min once a day in PBS
for 3 days. Next, the adherent cells were fixed with 2.5% (v/v) glutaraldehyde for 1h,
washed with PBS for 3 times, gradient ethanol dehydrated and observed by SEM after
6
drying. The cellular sample were also fixed with 4% paraformaldehyde, cellular actinView Article Online
DOI: 10.1039/C9BM01045A
and nuclei were stained with Actin-Tracker (Actin-Tracker Green Kit, Beyotime, China)
and Hoechst (Hoechst 33342, Sigma, America). The observation was performed on a
CLSM. To study the cell viability, 3T3 and JB6 cells were seeded in 24-well plates (1
× 104 cells per well) and cultured for 12 h. Then, four groups were set: control, PFMP,
30PFMP, 30PFMP-D after DD. Corresponding membranes were added to the wells,
the cells were cultured further for 1, 3, 5 days. Cell viability was measured using the

CCK-8 assay.
2.11. In Vivo Wound Healing Evaluation
The C57BL/6 mice (female, 7-8 weeks) were randomly divided into four groups (n=5
per group): control, PFMP, 30PFMP, 30PFMP-D after DD. The hair of the mouse's
hind part was shaved beforehand. A circular full-thickness skin wound with diameter
10 mm was created and covered with the corresponding membrane before shielded with
Tegaderm. The mice of control group were only covered with Tegaderm. The wounds
of mice were photographed every 4 days, the wound area was measured by Image-pro
plus. The relative wound area was calculated by: relative wound area (%) = (the wound
area on days t/ the wound area on day 0) × 100% (t = 0, 4, 8, 12). On day 15, the whole
wound tissue with a margin of around ambient unwounded skin was excised, all
samples were fixed in 4% paraformaldehyde solution, gradient ethanol dehydrated,
embedded in paraffin, and sectioned into 5 μm thick slices. The sections were
immunofluorescence staining with anti-CD31 antibody simultaneous staining of nuclei
with DAPI for analysis of angiogenesis in the wound bed.
3. Results and discussion
3.1. Preparation of Porous Fiber Membrane
Applying the breath figure mechanism [31, 32] to electrospinning technology is a

simple and effective method to obtain fibers with porous structure. The schematic
diagram demonstrating the formation of pores on fiber surface is shown in Fig. 1a. The
volatility of solvent and the humidity of environment during the electrospinning process
are two important factors influencing the surface structure of the fibers. When the
polymer is dissolved in a volatile solvent and electrospinning is carried out in a humid
environment, the evaporative cooling causes moisture to condense and form water
droplets on the surface of the fiber. After the evaporation of solvent and water, the
imprint of the water droplets leaves on the surface of the fibers, thus the porous structure
was obtained [33]. In addition, the properties of polymer can also affect the formation
of pores. Therefore, these three factors have been explored during the preparing process
of porous fiber membrane. The porous fiber membrane was named as PFM, and as
control, the smooth fiber membrane was named as SFM. PLA and PCL are both
biocompatibility and biodegradability polymers have been widely adopted for
biomedical applications. In comparison, PLA has relatively high mechanical properties
7
while PCL is more flexible [34]. In this study, the PLA/PCL blends were used DOI: to retain View Article Online
10.1039/C9BM01045A
the merits of both polymers. The volatile solvent DCM was chosen as the solvent. Due
to the poor conductivity of DCM, a small amount DMF was added to improve the
spinning efficiency.
The morphologies of PFMs prepared in different relative humidity (PLA/PCL= 3/1,
DCM/DFM=98/2) are shown in Fig. 1d-f. In 80% relative humidity, the porous
structure with uniform pore distribution could be clearly observed on the fiber surface.

And in 50% relative humidity, the fiber also had a distinct porous structure but the pore
size was slightly smaller. When the relative humidity dropped to 20%, the pores on
fiber surface were significantly reduced. Therefore, in order to obtain a porous structure,
the relative humidity of the environment should be kept above 50%. The morphologies
of PFMs with various PLA/PCL ratios of 3/1, 1/1, 1/3 (DCM/DFM=98/2, 50% relative
humidity) are shown in Fig. 1g-i. The effect of polymer on pore formation was
relatively small, but there was a better porous structure when the PLA ratio was higher.
Finally, the effect of solvent on the morphology of the fiber can be seen from Fig. 1j-l.
As the mixed solvent ratios of DCM/DFM were respectively 98/2, 95/5 and 90/10
(PLA/PCL= 3/1, 50% relative humidity). The pores on the fiber surface significantly
reduced when the proportion of DMF was increased. When the DMF ratio exceeds 10%,
the obtained fibers were almost smooth. So the DMF proportion must be controlled in
a very small range, too much DMF would affect the formation of pores. In summary,
optimal pore forming conditions are polymer PLA/PCL ratio in 3/1, mixed solvent
DCM/DFM ratio in 98/2 and electrospinning environment relative humidity higher than
50%. The porous structure formed under this condition is uniform and dense (Fig. 1b).
The pore size statistical results were fitted with Gaussian distribution (Fig. 1c). In the
experiment below, all PFMs were prepared under the obtained optimal conditions.
3.2. Preparation and Characterization of CuS Incorporated PNM
The CuS nanoparticles (Fig. 2a) were synthesized according to the previously
reported method [28]. The X-ray diffraction(XRD) pattern (Fig. 2b) of the CuS
nanoparticle matched well with the standard CuS phase (JCPDS card no. 6-0464). CuS
nanoparticles were incorporated into the porous fiber by simply premixed with the
spinning solution. The PNMs incorporated with 0%, 15%, 30%, 45% (m/m) CuS
nanoparticles (Fig. S1) were respectively named as 0PFM, 15PFM, 30PFM, 45PFM.
And 15PFM, 30PFM, 45PFM (Fig. 2d-f) have the similar porous structure to 0PFM
(Fig. 2c). That to say, the incorporating of CuS nanoparticles did not affect the pores
formation. But the distribution of CuS nanoparticles on the fiber membrane was
different. For 15PFM (Fig. 2l, m), most particles were embedded inside the fiber,
external appearance of the fiber was similar to 0PFM (Fig. 2j, k). While, some particles
on 30PFM were embedded on the fiber surface (Fig. 2n, o) besides the embedded inside
ones. For 45PFM, some particles were agglomerated (Fig. 2p, q), partial particles just
adhered on the fiber surface and were easy to fall off. Therefore, the amount of
incorporated CuS should be below 30%. Energy-dispersive spectrometry (EDS)
element mapping of 30PFM shows that the Cu and S elements (Fig. 2g, h) were
8
uniformly distributed within the fiber. The characteristic peaks of Cu and S in DOI:
the 10.1039/C9BM01045A
EDSView Article Online
spectrum (Fig. 2i) further confirmed the incorporating of CuS nanoparticles.
3.3. Preparation and Characterization of Drug Loaded PNM
Drug loading was achieved by simply immersing the fiber membrane in DOX
solution. Then a layer of PVA was coated on the fiber surface, which can improve the

fiber hydrophilicity and prevent the sudden release of the drug to a certain extent (Fig.
3a). The PVA coated SFM, PFM and 30PFM were respectively named as SFMP, PFMP
and 30PFMP, the corresponding drug-loaded and PVA coated membranes were
respectively named as SFMP-D, PFMP-D and 30PFMP-D. The morphology of
30PFMP is shown in Fig. S2 and the membrane still retained the fiber morphology.
Infrared spectra (Fig. 3b) of 30PFM and 30PFMP prove that PVA was successfully
coated on the fiber surface. As the absorption peak around 3300 cm-1 attributed to –OH
stretching vibration can be clearly observed on 30PFMP spectrum while not on 30PFM
spectrum. Contact angle tests of SFM, PFM, 30PFM and 30PFMP (Fig. 3c) were
conducted to study the change of fiber membrane wettability. The contact angle on
SFM was 125.7° while on PFM was 131.1°. This can be attributed to the fact that the
porous structure has a rougher surface and a larger surface energy. Thus the contact
angle was slightly increased. The contact angle of 30PFM was 115.6°, the addition of
CuS increased the hydrophilicity of the membrane. For 30PFMP, since the fiber was
coated with PVA, the membrane could be completely wetted with a contact angle of
0°.
The drug loading capacities of SFM, PFM and 30PFM were compared (Fig. 3d).
SFM has a loading capacity of 3.11 μg mg-1, while the PFM has a loading capacity of
9.49 μg mg-1. The rougher surface and larger specific surface area of the porous fiber
make it has a larger drug loading capacity. 30PFMP has a loading capacity of 10.41 μg
mg-1, even larger than PFM, this may be due to the adsorption of some drugs on CuS
nanoparticles embedded on the fiber surface. The CuS ultraviolet-visible absorption
spectroscopy is shown in Fig. S3. According to it, a 980 nm NIR laser was selected as
external light source that causes photothermal effects. The drug-loaded fiber
membranes release the drug slowly at body temperature (37°C). When exposed to 980
nm NIR laser, the temperature increase of the membrane accelerated the diffusion of
the drug, thus the drug released fast (Fig. S4). Under 980nm laser, the release rate of
the drug on 30PFMP reached 92.8% on day 7. This means the drug loaded on the porous
fiber can be almost completely released. A NIR-triggered drug release was investigated
in PBS (Fig. 3e), after 4 cycles “on and off” laser irradiation (0.5 W cm-2), the
percentage of released DOX of 30PFMP-D reached 37.01%. Since no CuS was
incorporated, the solution was hardly heated when the PFM was irradiated, the DOX
release percentage was only 16.45%. CuS nanoparticles successfully endowed the
composite porous fiber membrane photothermal property. When the fiber membrane
was irradiated by NIR, CuS increased the temperature of the fiber membrane by
photothermal conversion. The heat generated by it can accelerate the movement of the
drug molecules and enhance the permeability of the PVA on the surface of the fiber,
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thereby promoting the diffusion of the drug and increasing the release rate of the
DOI: drug.
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10.1039/C9BM01045A
The drug release from the membrane could be controlled by the NIR laser. As some
recent studies[29, 30] have confirmed that the copper ion can promote wound healing.
The Cu ion release behaviors from PFMP, 30PFMP and 30PFMP-D were investigated
(Fig. 3f). The membranes were immersed in PBS and Cu ion concentrations were
detected for 6 days. There was no Cu ion release in the PFMP group during the test
period. For 30PFMP and 30PFMP-D groups, the Cu ions were continuously released

and the concentration respectively reached 1.27 μg ml-1 and 1.20 μg ml-1 on the day 6.
3.4. Photothermal Performance of CuS Incorporated Membranes
To study the photothermal performance of the CuS incorporated membranes. PFMP,

15PFMP, 30PFMP and 45PFMP were irradiated by 980nm laser (0.5 W cm-2) for 120s
under dry condition (Fig. 4c) and for 10 min under wet condition (Fig. 4d), temperature
change was recorded in real time. The temperatures of 15PFMP, 30PFMP and 45PFMP
all increased rapidly and reached a stable state of 51.8°C, 57.3°C, 64.4°C within 40s in
dry environment. As control, the final temperature of PFMP was 32.1℃. In wet state,
the temperature rise rates of 15PFMP, 30PFMP and 45PFMP were relatively slow, and
when the temperature was stable, they reached 47.6°C, 52.0°C and 56.3°C within
10min respectively, and the temperature of PFMP was only 32.1℃. The thermal
infrared images recording the heating process of PFMP and 30PFMP in dry (Fig. 4a)
and wet (Fig. 4b) conditions further proved the good photothermal property of 30PFMP.
The effect of different power densities on the temperature rise was also explored (Fig.
S5). The temperature of 30PFMP could be controlled from room temperature to 61.1℃
by adjusting the power densities of the laser. Temperature elevation of 30PFMP over
five ON and OFF cycles of 980 nm laser irradiation (0.7 W cm-2) is shown in Fig. 4e.
The temperature elevations for the 4 cycles of irradiation could increase almost
identical from 26°C to about 67°C, indicating that 30PFMP has a good NIR
photostability which is beneficial to repeated photothermal therapy in clinical
treatments. 30PFMP can meet the temperature requirement for photothermal therapy
and the CuS nanoparticles in 30PFMP were not easy to fall off compared with 45PFMP.
Combined with the discussion above, 30PFMP can effectively load chemotherapeutic
drugs and control the drug release. At the same time, the drugs would have deeper
penetration depth and better therapeutic effect due to the increase of temperature.
Therefore, 30PFMP-D is the suitable choice for synergistic therapy.
3.5. In vitro anticancer performance
The in vitro anticancer efficiency was evaluated using calcein-AM/propidium iodide

(PI) (live/dead) staining coupled with the CCK-8 assay. In this test, six groups of
control, 30PFMP, 30PFMP-D, 30PFMP+Laser, 30SFMP+Laser and 30PFMP-
D+Laser were studied. As shown in cell viability assay (Fig. 5b), the 30PFMP has no
cytotoxicity against B16F10 cells, the 30PFMP-D and 30PFMP+Laser groups
representing the killing effect of chemotherapy (DOX) and photothermal effect on
10
melanoma cells had killed 31.8% and 49.7% of cells respectively. For 30PFMP- View Article Online
DOI: 10.1039/C9BM01045A
D+Laser group, the drugs and hyperthermia simultaneously work, the killing efficiency
could reach 76%. As the loading capacity of smooth fiber is much smaller than porous
fiber, the 30SFMP-D+Laser group had a relatively poor performance than 30PFMP-
D+Laser group, about 59.6% of cells were killed. The live/dead staining analysis was
consistent with the CCK-8 results (Fig. 5a). Relative cell viability of corresponding
group with different irradiation times (c) and different irradiation duration (d) of 980

nm laser are shown in Fig. 5c and Fig. 5d. The cell killing effects were all enhanced for
30PFMP, 30SFMP, 30PFMP-D with the increase of irradiation duration and the
irradiation times, but 30PFMP-D always has the best performance under the same
conditions. The PFMP-D was unaffected by irradiation duration and times.
3.6. In Vivo Antitumor Treatment

Encouraged by the outstanding in vitro anticancer performance, the tumor

therapeutic efficiency was further explored in vivo. The B16F10 tumor-bearing mice
were randomized into 6 groups: Control, 30PFMP, 30PFMP-D, 30PFMP+Laser,
30SFMP-D+Laser, 30PFMP-D+Laser. The tumors of mice in each group were covered
with corresponding membrane. For groups need laser treated, the membrane was
irradiated by 980nm laser (0.5 W cm-2) once a day for 3 days and 15min each time.
Thermal infrared images (Fig. 6a) of tumor-bearing mice in 30SFMP-D+Laser and
30PFMP-D+Laser groups were used to record the heating process. The temperature of
both 30SFMP-D+Laser and 30PFMP-D+Laser groups increased rapidly to 50°C. As
control, the temperature increased only to about 40°C for the mice covered with PFMP.
It can be found that on tumor growth curves (Fig. 6b), the tumors of control and
30PFMP groups had a very fast growth rate. The tumors of 30PFMP-D group were
slightly inhibited, the treatment effect of DOX on melanoma was no significant. A little
more pronounced inhibition of tumors was found in 30PFMP+Laser group, the
hyperthermia could suppress the tumor growth more effective than drugs on Melanoma.
Combining chemotherapy with photothermal therapy, the 30SFMP-D+Laser and
30PFMP-D+Laser groups significantly inhibited tumor growth. Since the porous fiber
loaded more drug than the smooth fiber, the therapeutic effect of 30PFMP-D+Laser
group was relatively better than 30SFMP-D+Laser group. The tumors of each group
were photographed (Fig. 6c) and weighed (Fig. 6f), 30PFMP-D+Laser group had the
minimized tumor volume on day 14 and lightest tumor weight, which is consistent with
the discussion above. The photographs of the tumor (Fig. 6b and Fig. S6) before (day
0) and after (day 14) various treatment can visually see the therapeutic effects of each
group. Meanwhile, no obvious abnormality was observed for body weight of the mice
in all six groups (Fig. 6e).
Furthermore, hematoxylin and eosin (H&E) staining, Ki67 immunohistochemical
staining and terminal-deoxynucleoitidyl transferase mediated nick end labeling
(TUNEL) staining of the tumor tissues (Fig. 7) on day 14 were performed, H&E
staining revealed that 30PFMP-D+Laser group treatment induced most tumor cells
destruction. Ki67 staining showed that the 30PFMP-D+Laser group treatment markedly
11
decreased the proliferation of Ki67-positive tumors cell expression. As demonstrated View Article Online
DOI: 10.1039/C9BM01045A
by TUNEL staining, 30PFMP-D+Laser group also have the highest TUNEL-positive
cells proportion, induce much more tumor cell apoptosis. It is obvious that the
synergistic chemo-photothermal therapy of 30PFMP-D shows the best therapeutic
effect on melanoma. As the high temperature can change the blood perfusion of
capillaries and accelerate drug release, thus increasing the concentration of drugs in the
tumor sites. The hyperthermia can also destroy the stability of tumor cells, increases

the permeability of cell membrane, and thus facilitates cellular uptake of drugs. In
addition, the sensitivity of drug-resistant cells to anticancer drugs has also been
enhanced. Therefore, the platform combining photothermal therapy and chemotherapy
have achieved very excellent therapeutic effect.
3.7. In Vivo Wound Healing Efficacy in Tumor-bearing Mice

It can be found that in Fig. 6b and Fig. S6, due to the influence of tumors, the skin
healing of the control, 30PFMP, 30PFMP-D, 30PFMP+Laser groups was hindered, the
skin around the tumor was thin and the skin structure was incomplete (Fig. S7). The
tumors of 30SFMP-D+Laser group were small and the skin wound is significantly
reduced, while the tumors of 30PFMP-D+Laser group were gradually disappeared
without recurrence and the skin almost completely healed. 30PFMP-D+Laser group
had the best anti-tumor effect and significantly accelerated wound healing in tumor-
bearing mice.
3.8. In Vitro Cell Adhesion and Proliferation
In addition to the treatment of tumors, the promotion of skin regeneration is another

important function of 30PFMP-D. To explore the biocompatibility and cell adhesion of
fiber membranes. The morphologies of 3T3 and JB6 cells on 30PFMP and 30PFMP-D
after drug delivery (DD) were investigated by SEM and CLSM after the cell seeding
on membrane after 24h (Fig. 8a). In SEM images, both 3T3 and JB6 cells on 30PFMP
and 30PFMP-D after DD exhibited with flattening morphology. The cells were closely
attached to the surface of the membrane and spread well. The cells also showed well
morphology in all CLSM fluorescence images. It indicates that the drug-loaded
30PFMP after release of drug still beneficial for cell adhesion. CCK-8 assay of 3T3
(Fig. b) and JB6 (Fig. c) showed that the proliferation rates of both kinds of cells
incubated with PFMP, 30PFMP and 30PFMP-D after DD were higher than that of
control. 30PFMP group had the best result, followed by 30PFMP-D after DD group.
This indicates that the 30PFMP-D after DD has almost no cytotoxicity. All membranes
in three groups have good biocompatibility.
3.9. In Vivo Wound Healing Efficacy
In order to exclude the negative effects of cancer on the skin wound healing, full
thickness cutaneous wound healing model was constructed on healthy C57BL/6 mice
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to study the wound healing efficiency of the following four groups: control,DOI: PFMP, View Article Online
10.1039/C9BM01045A
30PFMP, 30PFMP-D after DD. As shown in photographs at different time point (Fig.
9a), traces of wound bed closure (Fig. 9b) and wound healing efficiency (Fig. 9c), with
the number of days increased, the wound areas in all four groups became smaller. There
was no significant difference of the wound area on day 4. But on day 8 and day 12, it
can be clearly observed that the wound areas of PFMP, 30PFMP and 30PFMP-D after
DD groups were smaller than that of control group. This is because that the fibrous

membrane covering on the skin defect provided mechanical support and structural cues
for wound healing. The remaining wound areas of control, PFMP, 30PFMP, 30PFMP-
D after DD were respectively 5.3%, 3.4%, 1.7%, 2.3% on day 12. 30PFMP and
30PFMP-D after DD groups had even better results than PFMP group. H&E staining
(Fig. 9d) and CD31 staining (Fig. 9e) of the healed skin on day 12 showed that the
30PFMP and 30PFMP-D after DD groups had the larger thickness of granulation tissue
and more CD31 positive cells in the wound bed. It means the blood vessel densities of
these two groups were higher and the capillary formation at wound sites was
significantly enhanced. This may be related to the release of therapeutic copper ions
from the fiber membrane of 30PFMP and 30PFMP-D after DD groups. The copper ions
artificially mimicked the hypoxia by stabilizing the expression of hypoxia-inducible
factor (HIF-1a), thereby upregulating vascular endothelial growth factor (VEGF)
express [35]. What is more, Cu2+ is also known to affect the activity of crucial protein
during wound healing processes. The 30PFMP-D after DD group had the similar result
to the 30PFMP group, it proved that the membrance performance is not affected after
the drug delivery stage, and can still effectively promotes wound healing.
4. Conclusion
In conclusion, we have developed a dual-functional porous fiber membrane for

melanoma treatment and skin wound healing. Uniform pore structure on the fiber
surface was obtained and significantly improved drug loading capacity. By
incorporating CuS nanoparticles, the fiber membrane had an excellent photothermal
effect, and the drug release rate can be controlled by temperature changes caused by
NIR irradiation. Thus, the membrane has excellent chemical-photothermal synergistic
therapeutic effects on melanoma, exceeding single chemotherapy or photothermal
therapy. Simultaneously, the porous fiber membrane could effectively promote the
cutaneous wound healing by providing mechanical support, structural cues and
releasing of therapeutic copper ions. This work broadens the applications of electrospun
scaffold drug delivery system and provides new possibilities for the development of
multifunctional scafflods for cancer local treatment and tumor resection postoperative
care.
Conflicts of interest
The authors declare no conﬂict of interest.
13
Acknowledgements View Article Online

DOI: 10.1039/C9BM01045A
This work was supported by Beijing Natural Science Foundation (2172039), Beijing
Science Technology New Star Cross Subject (2018019), National Key Research and
Development Program of China (2018YFD0401302), and the Fundamental Research
Funds for the Central Universities and USTB.
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DOI: 10.1039/C9BM01045A

Fig. 1. Schematic diagram(a) illustrating the formation mechanism of pores on the

surface of electrospun fibers. Porous fibers under optimal conditions (b) and
corresponding pore size distribution and Gaussian fitting (c). SEM image of surface
morphology of individual fibers under various relative humidity 80% (d), 50% (e) 20%
(f), various PLA/PCL polymer ratio 3/1 (g), 1:1 (h), 1:3 (i) and various DCM/DMF
ratio of mixed solvent 98/2 (j), 95:5 (k), 90:10 (l).
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DOI: 10.1039/C9BM01045A

Fig. 2. TEM image (a) and X-ray diffraction pattern (b) of CuS nanoparticles to
characterize the morphology and phase. SEM images of 0PFM (c), 15PFM (d), 30PFM
(e), 45PFM (f), the EDS elemental mappings of 30PFM for Cu (g) and S (h) and EDS
spectrum (i) of 30PFM. SEM and TEM images showing the typical form of CuS
nanoparticle embedded in 0PFM (j, k), 15PFM (l, m), 30PFM (n, o), 45PFM (p, q).
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Fig. 3. Schematic diagram (a) illustrating the drug loading process on 30PFM and its
controlled release mechanism. Infrared spectroscopy (b) of 30PFM and 30PFMP prove
that PVA was successfully coated on the fiber surface. Contact angle analyses (c) to
study the hydrophilicity of SFM, PFM, 30PFM and 30PFMP. Drug loading capacity
(d) of SFM, PFM and 30PFM. NIR-triggered drug release profile (e) of 30PFMP-D.
The release profiles(f) of Cu ions from PFMP, 30PFMP and 30PFMP-D after DD in
PBS for 6 days at 37℃.
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DOI: 10.1039/C9BM01045A

Fig. 4. The thermal infrared images of PFMP and 30PFMP irradiated by 980nm laser
(0.5 W cm-2) in real time under dry (a) and wet (b) conditions. The photothermal heating
curves of PFMP, 15PFMP, 30PFMP and 45PFMP irradiated by 980nm laser (0.5 W
cm-2) for 120s under dry condition (c) and for 10 min under wet condition (d). (e)
Temperature elevation of 30PFMP over five ON and OFF cycles of 980 nm laser
irradiation (0.5 W cm-2).
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Fig. 5. In vitro anticancer efficiency. (a) live and dead staining images (green: live cells;
red: dead cells) of B16F10 cells for 30PFMP, 30PFMP-D without 980nm laser
Irradiation and 30PFMP, 30SFMP-D, 30PFMP-D with 980nm laser Irradiation and
corresponding relative cell viability (b). Relative cell viability of the 980nm laser
treated groups with different irradiation times (c) and different irradiation duration (d).
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DOI: 10.1039/C9BM01045A

Fig. 6. In vivo anticancer efficiency. (a) thermal infrared images of B16F10 tumor-
bearing mice covered with PFMP(control), 30SFMP-D and 30PMFP-D when exposed
to the 980 nm laser irradiation. Photographs of melanoma-bearing mice (b) on day 0
and day 14 after various treatment and the excised tumors (c) on day 14. Growth curves
of tumor (d) and body weight (e) of melanoma-bearing mice of different groups in 14
days. Tumor weight (e) on day 14 of different groups.
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Fig. 7. Hexatoxylin and eosin (H&E) staining, Ki67 immunohistochemical staining,

and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL)
staining (green: apoptotic cell; blue: nucleus) images of the tumor tissues in different
groups.
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Fig. 8. In vitro cell adhesion and proliferation on porous fiber membrane. (a)SEM
images and CLSM fluorescence images of 3T3 and JB6 cells on 30PFMP and 30PFMP-
D after DD. (b) Cell proliferation of 3T3 cell (b) and JB6 cell (c) on PFMP, 30PFMP
and 30PFMP-D after drug delivery for 1, 3 and 5 days.
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Fig. 9. In vivo wound healing. (a) Photographs of skin wound on days 0, 4, 8 and 12.
Traces of wound bed closure (b) and the statistical analysis of wound area to show the
healing efficiency (c) of control, PFMP, 30PFMP and 30PFMP-D after DD groups.
Immunofluorescence staining (red: CD31, blue: nuclei) and H&E staining images (d)
of wound healing skin 12 days after treatment. Quantitative analysis of CD31-positive
blood vessels (e) of the wound healing skin.
24
A controllable local drug delivery system can effectively inhibit melanoma growth withView Article Online
DOI: 10.1039/C9BM01045A
chemo-photothermal synergistic therapy and accelerate wound healing.


A Controllable Local Drug Delivery System Based On Porous Fibers For Synergistic Treatment of Melanoma and Promoting Wound Healing

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A Controllable Local Drug Delivery System Based On Porous Fibers For Synergistic Treatment of Melanoma and Promoting Wound Healing

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for Synergistic Treatment of Melanoma and Promoting Wound

Biomaterials Science Accepted Manuscript

E-mail address: wyq_wen@ustb.edu.cn

Biomaterials Science Accepted Manuscript

Biomaterials Science Accepted Manuscript

2.2. Preparation and Conditional Exploration of Porous PLA/PCL Fiber Membrane

2.3. Synthesis of CuS NPs

Biomaterials Science Accepted Manuscript

2.4. Preparation of CuS Incorporated PLA/PCL PNMs

2.5. Drugs Loading and NIR-Triggered Release in Vitro

SNM, PNM and 30PNM were respectively immersed in 10 ml doxorubicin

2.6. Material Characterization

The morphologies and elemental distribution were observed by scanning electron

Biomaterials Science Accepted Manuscript

2.7. Photothermal Performance of PNMPs

The photothermal performance of PFMP, 15PFMP, 30PFMP, 45PFMP which

2.8. In Vitro Anticancer Effect

Mouse B16F10 melanoma cells were cultured in Dulbecco’s modified Eagle’s

2.9. In Vivo Antitumor Efficacy and Wound Healing Evaluation

Biomaterials Science Accepted Manuscript

2.10 In Vitro Cell Adhesion and Proliferation

Biomaterials Science Accepted Manuscript

2.11. In Vivo Wound Healing Evaluation

3. Results and discussion

3.1. Preparation of Porous Fiber Membrane

Applying the breath figure mechanism [31, 32] to electrospinning technology is a

Biomaterials Science Accepted Manuscript

3.2. Preparation and Characterization of CuS Incorporated PNM

3.3. Preparation and Characterization of Drug Loaded PNM

Biomaterials Science Accepted Manuscript

Biomaterials Science Accepted Manuscript

3.4. Photothermal Performance of CuS Incorporated Membranes

To study the photothermal performance of the CuS incorporated membranes. PFMP,

3.5. In vitro anticancer performance

The in vitro anticancer efficiency was evaluated using calcein-AM/propidium iodide

Biomaterials Science Accepted Manuscript

3.6. In Vivo Antitumor Treatment

Encouraged by the outstanding in vitro anticancer performance, the tumor

Biomaterials Science Accepted Manuscript

3.7. In Vivo Wound Healing Efficacy in Tumor-bearing Mice

3.8. In Vitro Cell Adhesion and Proliferation

In addition to the treatment of tumors, the promotion of skin regeneration is another

3.9. In Vivo Wound Healing Efficacy

Biomaterials Science Accepted Manuscript

In conclusion, we have developed a dual-functional porous fiber membrane for

Acknowledgements View Article Online

Biomaterials Science Accepted Manuscript

Biomaterials Science Accepted Manuscript

Liang and C. Li, J. Am. Chem. Soc., 2010, 132(43), 15351-15358.

View Article Online

Biomaterials Science Accepted Manuscript

Fig. 1. Schematic diagram(a) illustrating the formation mechanism of pores on the

View Article Online

Biomaterials Science Accepted Manuscript

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Biomaterials Science Accepted Manuscript

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Biomaterials Science Accepted Manuscript

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