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Nano Res

1

Multifunctional electrospinning composite fibers for

orthotopic cancer treatment in vivo

Yinyin Chen1,2, Shi Liu2, †, Zhiyao Hou1, Pingan Ma1, Dongmei Yang1,2, Chunxia Li 1 () and Jun

Lin1()

Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-014-0701-y

http://www.thenanoresearch.com on December 23 2014

© Tsinghua University Press 2014

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Nano Research

DOI 10.1007/s12274-014-0701-y

Multifunctional Electrospinning Composite Fibers for

Orthotopic Cancer Treatment in Vivo



Yinyin Chen1,2, Shi Liu2, , Zhiyao Hou1, Pingan Ma1,

Dongmei Yang1,2, Chunxia Li 1*, and Jun Lin1*

1State Key Laboratory of Rare Earth Resource

Utilization, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130024, P. R.

China

2 University of the Chinese Academy of Sciences Beijing

100049, P.R. China

State Key Laboratory of Polymer Physics and Chemistry,

Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences

A multifunctional dual drug carrier platform

DOX-NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-PEG@MC-PG was

successfully assembled via electrospinning process. The resultant

multifunctional spinning pieces can be implanted directly to the tumor

site of mice by surgical procedures to fulfill the orthotopic

chemotherapy by the controlled release of DOX from mesoporous

SiO2 and the upconversion fluorescence/magnetic resonance dual

model imaging through NaGdF4:Yb/Er@NaGdF4:Yb embedded in

MC/UCNPS/DOX in vivo.



Yinyin Chen1,2, Shi Liu2, †, Zhiyao Hou1, Pingan Ma1, Dongmei Yang1,2, Chunxia Li 1 () and Jun Lin1()

Received: day month year

Revised: day month year

Accepted: day month year

(automatically inserted by

the publisher)

© Tsinghua University Press

and Springer-Verlag Berlin

Heidelberg 2014

KEYWORDS

Electrospinning

Orthotopic, Treatment,

Controlled Release,

Multiple Structure

ABSTRACT

A multifunctional dual drug carrier platform was successfully assembled. The

antitumor drug doxorubicin (DOX) loaded core-shell structured

NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-polyethylene glycol (abbreviated as UCNPS)

nanoparticles were incorporated into antiphlogistic drug indomethacin (MC)

loaded poly(ε-caprolactone) (PCL) and galatin to form nanofibrous fabrics (labeled

as MC/UCNPS/DOX) via electrospinning process. The resultant multifunctional

spinning pieces can be implanted directly to the tumor site of mice by surgical

procedures to fulfill the orthotopic chemotherapy by the controlled release of DOX

from mesoporous SiO2 and the upconversion fluorescence/magnetic resonance dual

model imaging through NaGdF4:Yb/Er@NaGdF4:Yb embedded in

MC/UCNPS/DOX in vivo.

1. Introduction

Both unresectable tumors for example hepatocellular

carcinoma and metastases cancer such as breast

cancer, renal carcinoma, lung cancer and so on

represent a major clinical problem owing to the poor

prognosis. There are about 50–80% of patients

experiencing recurrence by 5 years after resection,

partly resulting from invisible intrahepatic

Nano Research

DOI (automatically inserted by the publisher)

Review Article/Research Article Please choose one

Address correspondence to Jun Lin. [email protected]; Chunxia Li. [email protected]

| www.editorialmanager.com/nare/default.asp

2 Nano Res.

metastases during resection [1]. For the treatment of

unresectable cancer or for the prevention of

post-surgery tumor recurrence, chemotherapy will be

a good choice. The nonspecific systematic

distribution of the antitumor drugs is one of the main

disadvantages of the conventional tumor

chemotherapy [2-4]. In this case, orthotopic treatment

is an inevitable and promising approach. Although

many targeting therapeutic strategies have been

developed, due to the difficulty to transport

effectively the chemotherapy drugs to the specific

location in the context of multiple in vivo

physiological barriers [5, 6], the management of

malignant cancers still remains clinical challenge.

Thus, it will be necessary to prolong blood

circulation of antitumor drug. Owing to the excellent

character of mesoporous SiO2, such as good

biocompatibility, large specific surface area, tunable

mesoporous structure, and facile surface

functionalization and so on, it has recently acted as a

potential anticancer therapy, from which the

prolonged drug release with tunable drug release

kinetics could be achieved. Moreover, mesoporous

SiO2 could enhance the dissolution of the poorly

water-soluble drugs and increase their bioavailability,

and mesoporous SiO2 with small sizes preferably

accumulate at tumor sites caused by the enhanced

permeability and retention (EPR) effect [7, 8].

Electrospinning is a cutting edge technology for

producing continuous polymer fibers that has

recently attracted attention in the field of drug

delivery [9, 10]. Owing to their unique characteristics

such as extremely high surface area and excellent

pore interconnectivity, electrospun polymeric fibers

are particularly attractive for carriers for a series of

drugs and even can be engineered to smart delivery

drug in a controlled fashion [11-16]. More importantly,

the electrospun fibers as a kind of implant materials

can be exploited to site-specific delivery of drugs to

the body as well as wound healing or surgery

treatment.

Molecular imaging techniques, such as magnetic

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

3 Nano Res.

resonance imaging (MRI) [17, 18], X-ray computed

tomography (CT), upconvension fluorescence

microscopy [19-22], and positron emission tomography

(PET) play an important role in medicine and

biomedical research [23, 24]. The information obtained

from single modal molecular imaging cannot satisfy

the higher requirements on the efficiency and

accuracy for clinical diagnosis and medical research

[25, 26]. Thus, multimodality imaging will provide more

complementary, effective and accurate information

on the physical anatomical structure and the

physiological function for diagnosis and treatment.

Resulting from their special 4f electron structure and

rich optical-magnetic properties [27-29],

lanthanide-based nano-probes have attracted

increasing attention in multimodal molecular

imaging. In particular, due to the existence of seven

unpaired electrons in 4f orbit of Gd3+ ions,

upconversion nanoparticles containing Gd3+ ions can

exhibit fluorescent and magnetic properties.

Therefore, such nanoparticle can be regarded as a

multimodal imaging biological probe for

simultaneous UCL and MRI.

So in this work, we put forward a

multifunctional anticancer drug carrier platform, in

which antitumor drug doxorubicin (DOX) loaded

core-shell structured

NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-polyethylene

glycol (abbreviated as UCNPS) nanoparticles were

incorporated into antiphlogistic drug indomethacin

(MC) loaded poly(ε-caprolactone) (PCL) and galatin

to form nanofibrous fabrics (labeled as

MC/UCNPS/DOX) via electrospinning process. The

resultant multifunctional spinning pieces can be

implanted directly to the tumor site of mice by

surgical procedures to fulfill the orthotopic

chemotherapy by the controlled release of DOX from

mesoporous SiO2 and the upconversion

fluorescence/magnetic resonance dual model

imaging through NaGdF4:Yb/Er@NaGdF4:Yb

embedded in MC/UCNPS/DOX in vivo.

2. Materials and methods


4 Nano Res.

2.1. Materials

The rare earth chloride RECl3·6H2O (99.99%, RE=Y,

Yb and Er), oleic acid (90%, technical grade),

Octadecene (90%, technical grade),

Poly(ε-caprolactone) (PCL, Mw = 70,000-90,000),

Indomethacin (MC, 99%), Gelatin (pharmaceutical

grade) and 2,2,2-Trifluorothanol (TFE, 99.8%,

molecular biology grade) were purchased from

Aldrich. 2-[methoxy-(polyethyleneoxy)

propyl]trimethoxysilane (PEG500-silane, Mw = 460-590,

tech-90) was purchased from Gelest. Doxorubicin

hydrochloride (DOX) was purchased from Nanjing

Duodian Chemical Limited Company (China). Other

reagents including cetyltrimethylammonium

bromide (CTAB, ≥99%), tetraethylorthosilicate

(TEOS), ammonium fluoride (NH4F), sodium

hydroxide (NaOH, ≥98%) and ammonium nitrate

(NH4NO3, ≥99.0%) were purchased from Beijing Yili

Fine Chemical Regent Company (China). All the

chemical reagents were used as received without

further purification.

2.2. Preparation of

NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-PEG

(UCNP@mSiO2-PEG) nanoparticles

The preparation of NaGdF4:Yb/Er@NaGdF4:Yb

(UCNP for short) nanocrystals [Gd:Yb:Er=80:18:2

(mol ratio) in core and Gd:Yb=80:20 (mol ratio) in

shell] followed by mesoporous silica coating and

PEG modification was carried out according to our

previous approach [30-32]. The as-obtained

nanomaterials were labeled as UCNP@mSiO2-PEG.

2.3. Fabrication of drug delivery systems

2.3.1. DOX-loaded UCNP@mSiO2-PEG nanoparticles

10 mg of UCNPS sample dispersing in 2 mL of water

was mixed with 2 mL of DOX aqueous solution (1

mg/mL). After stirred for 24 h under dark conditions,

the DOX-loaded sample was collected by

centrifugation and denoted as UCNPS/DOX. The

as-obtained nanomaterials were denoted as

DOX-UCNP@mSiO2-PEG. To evaluate the

DOX-loading efficiency, the residual DOX content

(RDOX) in the supernatant and washed solutions was


5 Nano Res.

obtained by UV-Vis measurement at a wavelength of

480 nm [33]. UV-Vis measurement of DOX at a

wavelength of 480 nm was used to evaluate the

DOX-loading efficiency by formula: [(ODOX －

RDOX)/ODOX]×100%, in which ODOX and RDOX is the

original DOX content and the residual DOX content

in the supernatant, respectively. The loading capacity

of DOX is 10.5%. Then, UCNPS/DOX samples were

immersed in 2 mL pH = 7.4 and 6.2 phosphoric acidic

buffer solutions (PBS) at 37 °C with gentle shaking.

At predetermined time intervals, PBS was taken by

centrifugation and replaced with an equal volume of

fresh PBS. The amount of released DOX in the

supernatant solutions was measured by UV-Vis

spectrophotometer at a wavelength of 480 nm.

2.3.2. DOX-loaded composite fibers (CFs)

Gelatin and PCL were dissolved separately in 2.5 mL

transparent DOX-TFE solution (1 mg/mL) under

stirring. The mass ratio of Gelatin and PCL was 1:1.

When Gelatin and PCL dissolved completely in

DOX-TFE solution, Gelatin and PCL were mixed

isometricly together and continued to stir for 30 min

to obtain a homogeneous precursor sol for further

electrospinning. The parameters adjustment of the

spinning equipment was based on our previous

method [11, 12]. The distance between the spinneret (a

metallic needle) and collector (a grounded conductor)

was fixed at 10 cm and the high-voltage supply was

maintained at 10 kV. The spinning rate was

controlled at 1.0 mL/h by a syringe pump

(TJ-3A/W0109-1B, Baoding Longer Precision Pump

Co., Ltd, China). The DOX-PG (labeled as DOX) CFs

was fabricated, dried and stored at 4 oC for further

using. Then, the DOX release experiments from

DOX-UCNP@mSiO2-PEG samples were similar to the

above procedures.

2.3.3. UCNP@mSiO2-PEG-loaded CFs

Gelatin and PCL were each dissolved separately in

2.5 mL UCNP@mSiO2-PEG-TFE solution (4 mg/mL,

20 mg UCNP@mSiO2-PEG dissolving in 5 mL TFE)

under stirring condition. The mass ratio of Gelatin

and PCL was 1:1. When Gelatin and PCL dissolved


6 Nano Res.

completely in UCNP@mSiO2-PEG-TFE solution,

Gelatin and PCL were mixed isometricly together

and continued to stir for 30 min to obtain a

homogeneous precursor sol for further

electrospinning. The rest procedures for

UCNP@mSiO2-PEG@PG (labeled as UCNPS) CFs are

similar to those of DOX-loaded CFs.

2.3.4. DOX-loaded UCNP@mSiO2-PEG single drug

delivery system CFs

The DOX-loaded progress of UCNP@mSiO2-PEG was

implemented according to our previous operational

approach [11, 12]. 20 mg of UCNP@mSiO2-PEG was

dispersed and sonicated in 3 mL of DOX-TFE

solution (1 mg/mL). After stirred for 24 h under dark

and sealed conditions, the DOX-UCNP@mSiO2-PEG

nanoparticles were collected by centrifugation. These

DOX-loaded nanoparticles were dispersed and

sonicated in 5 mL TFE solution for 1 min, and then

0.25 g of PCL and 0.25 g of gelatin were added to the

suspension with continuous stirring for 3 h to form

electrospun precursor solution. The as-obtained

nanomaterials were denoted as

DOX-UCNP@mSiO2-PEG. Then the

DOX-UCNP@mSiO2-PEG nanoparticles were

dispersed and sonicated in 5 mL TFE solution to

obtain DOX-UCNP@mSiO2-PEG-TEF solution and

then Gelatin and PCL were added to above

suspension isometricly and continued to stir for 3 h



(DOX-UCNP@mSiO2-PEG)@PG (labeled as

UCNPS/DOX) CFs are similar to those of

DOX-loaded CFs.

2.3.5. DOX and MC co-loaded UCNP@mSiO2-PEG

dual drugs delivery system CFs

The DOX-loaded progress of UCNP@mSiO2-PEG was

implemented according to our previous operational

approach [11, 12]. 20 mg of UCNP@mSiO2-PEG was

dispersed and sonicated in 3 mL of DOX-TFE

solution (1 mg/mL). After stirred for 24 h under dark

and sealed conditions, the DOX-UCNP@mSiO2-PEG

nanoparticles were collected by centrifugation. These


7 Nano Res.

DOX-loaded nanoparticles were dispersed and

sonicated in 5 mL of MC-TFE solution (20 mg/mL, 20

mg MC dissolving in 1 mL TFE) for 1 min, and then

0.25 g of PCL and 0.25 g of gelatin were added to the

suspension with continuous stirring for 3 h to form

electrospun precursor solution. The as-obtained

nanomaterials were denoted as

DOX-UCNP@mSiO2-PEG. Then the

DOX-UCNP@mSiO2-PEG nanoparticles were

dispersed and sonicated in 5 mL of MC-TFE solution

(20 mg/mL, 20 mg MC dissolving in 1 mL TFE) to

obtain DOX-UCNP@mSiO2-PEG-TEF-MC solution

and then Gelatin and PCL were added to above

suspension isometricly and continued to stir for 3 h



(DOX-UCNP@mSiO2-PEG)@(MC-PG) (labeled as

MC/UCNPS/DOX) CFs are similar to those of

DOX-loaded CFs.

2.4. In vivo UCL imaging of composite fibers

Kunming mice were purchased from Changchun

Institute of Biological Products Co. Ltd and all

animal procedures were approved by the University

Animal Care and Use Committee. The tumors were

established by subcutaneous injection of mouse

hepatoma H22 cells as described previously [34].

Briefly, hepatoma H22 cells, kindly gifted by

Chemical biology laboratory of Changchun Institute

of Applied Chemistry Chinese Academy of Sciences

(Changchun, China), were suspended in

physiological saline and injected intraperitoneally

into the mice for serial subcultivation. The mice with

viable H22 ascites tumors were sacrificed, and the

ascites were withdrawn and diluted with

physiological saline to modulate the cell density at 1

×107 cells/mL. The ascites was injected

subcutaneously to each mouse at the left axilla at a

dose of around 0.01 mL/g body weight. The tumors

were allowed to grow for several days to reach the

size of around 200 mm3. MC/UCNPS/DOX CFs mat

(about 15000 mm2) of required sizes (60-100 mm2/mat,

8 pieces/mouse), flexibly resized according to the


8 Nano Res.

diameter of tumor nodules, was directly pasted on

the surface of the tumor of the tumor-bearing mice.

After 0 h, 24 h and 96 h, the UCL imaging

experiments were conducted by exposing the tumor

to a continuous semiconductor laser with an output

wavelength of 980 nm (1.2 W) and capturing

fluorescent signal by a camera.

2.5. In vivo MR imaging of composite fibers

MC/UCNPS/DOX CFs mat (about 15000 mm2) of

required sizes (60-100 mm2/mat, 8 pieces/mouse),

flexibly resized according to the diameter of tumor

nodules, was directly pasted on the surface of the

tumor of the tumor-bearing mice. After 2 days, MR

imaging studies were conducted on a 1.2 T clinical

MRI scanner [Atlas tong nuclear magnetic, shanghai,

China] equipped with a special coil designed for

small animal imaging.

2.6. In vivo therapy of composite fibers on

subcutaneous tumor model with composite fibers

When the size of tumors reach around 200 mm3, the

tumor-bearing mice were randomly divided into

groups with 5 animals in each group (n = 5). Then

DOX CFs mat, UCNPS CFs mat, UCNPS/DOX CFs

and MC/UCNPS/DOX CFs mat (about 15000 mm2) of

required sizes (60-100 mm2/mat, 8 pieces/mouse),

flexibly resized according to the diameter of tumor

nodules, was directly pasted on the surface of the

tumor (n = 5). The control group was without

administration. The body weights, tumor volumes,

and survival rate of animals were monitored every

other day after treatment. The length of the major

axis (longest diameter) and minor axis

(perpendicular to the major axis) of the tumor were

measured with a vernier caliper, and the tumor

volume was calculated as described previously. The

diameter of tumor was measured and the tumor

volume was calculated as described previously [35].

After 17 days treatment, all animals of each group

were euthanized to retrieve tumors and organs.

During the entire experiments, only a mouse in

control group died. To get a picture, we choose 4

tumors to take a photo after euthanizing all mice of


9 Nano Res.

each group. The excised tumors and organs were

washed by deionized water and then were fixed by

4% (weight) paraformaldehyde solution. The tissues

were processed routinely, and sections were stained

with H&E [34]. Blood of experimental group and

control group was collected based on our previous

method. The collected blood samples were sended

for blood chemistry tests and complete blood panel

analysis [36].

2.7. MC in MC/UCNPS/DOX CFs helps to heal the

wounds

The tumors were allowed to grow for several days to

reach the size of around 200 mm3. 9 mice were

randomly divided into three groups. Then

UCNPS/DOX CFs and MC/UCNPS/DOX CFs mat

(about 15000 mm2) of required sizes (60-100 mm2/mat,

8 pieces/mouse,1 mg MC), were directly pasted on

the surface of the tumor (n = 3). Then the incision was

sutured. After 4 h, the blood of two animals in each

group was collected for blood routine examination.

And the remaining mice was kept growing in order

to observe their incision healing.

2.8. Biodistribution and release of drug in vivo

The tumors were allowed to grow for several days to

reach the size of around 200 mm3.The tumor-bearing

30 female Kunming mice in a weight range of 20-25 g

(8-12 weeks old) were divided into 10 groups with 3

mice in per group. These mice were used for the

study of drug release profile and the biodistribution

of MC/UCNPS/DOX CFs in vivo. MC/UCNPS/DOX

CFs mat (about 15000 mm2) of required sizes (60-100

mm2/mat, 8 pieces/mouse), flexibly resized according

to the diameter of tumor nodules were directly

pasted on the tumor. The animals were sacrificed at

0.5 h, 2 h, 8 h, 12 h, 24 h and 2, 3, 5, 7 days after

fiber-mat implantation and samples of tumor, liver,

kidneys, spleen lungs, and heart were harvested after

the remaining spinning pieces were removed by

surgical operation. The excised tissues and organs

were imaged by fluorescent imaging system (CRI

Maestro 500 FL) to follow the release of DOX from

the fiber-mat and its biodistribution in mice. Fixed


10 Nano Res.

exposure time was adopted at all time points.

Semi-quantitative comparison between different

organs and different time intervals was also made by

means of commercial software (MaestroTM2.4).

2.9. Biodistribution measurement

The tumor-bearing 24 female Kunming mice in a

weight range of 20-25 g (8-12 weeks old) were

divided into 8 groups with 3 mice in per group.

These mice were implanted with MC/UCNPS/DOX

CFs mats. Then, the mice were sacrificed at 0 h, 0.5 h,

2 h, 8 h, 12 h, 24 h, 2 days and 7 days. Major organs

and tissues (tumor, heart, liver, spleen, lung, kidney)

were excised and collected after the remaining

spinning pieces were removed by surgical operation.

The excised organs and tissues wet weighed and

dissolved in digesting solutions (HNO3:H2O2 = 1:2 by

volume) [37]. The samples were heated at 70 oC for 4 h.

After cooling down to room temperature, the volume

of each sample solution was measured and

subsequently analyzed by ICP-AES to determine the

total amount of Gd3+ in each measured tissue. Three

animals per group were used in the biodistribution

measurement.

3. Results and discussion

3.1. Fabrication and characterization of the

materials

The working principle of our strategy is shown in

Scheme 1. Firstly, antitumor drug DOX delivery

carrier UCNP@mSiO2-PEG nanospheres were

fabricated according to a phase transfer assisted

surfactant-templating coating process reported

recently by us [30-32]. Subsequently, the as-obtained

DOX-UCNP@mSiO2-PEG were mixed with

electrospinning solution including PCL-gelatin (PG)

and anti-inflammatory MC so as to form dual

drugs-loaded multiple structure composite fibers

(DOX-NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-PEG)@(M

C-PG) (labeled as MC/UCNPS/DOX) via

electrospinning technique. The resultant

MC/UCNPS/DOX spinning piece can be implanted

directly to the tumor site of mice by surgical

procedures to fulfill the orthotopic efficient


11 Nano Res.

chemotherapy by the controlled release of DOX from

mesoporous SiO2 and the upconversion

fluorescence/magnetic resonance dual model

imaging through NaGdF4:Yb/Er@NaGdF4:Yb

embedded in MC/UCNPS/DOX in vivo.

Fig. 1 shows the morphology of the

nanoparticles at the different synthesis stages. The

typical TEM image (Fig. 1(a)) indicates that

cetyltrimethylammonium bromide (CTAB)-stabilized

NaGdF4:Yb/Er@NaGdF4:Yb nanoparticles (labeled as

UCNP) in aqueous solution has uniform shape with

mean diameter of 25 nm. The high-resolution TEM

image has revealed the obvious crystal lattices with

interplanar distance of 0.30 nm (Fig. 1(b)), which can

be assigned to (110) plane of β-NaGdF4. After

mesoporous silica coating and PEG modification, the

as-obtained UCNP@mSiO2-PEG takes on obvious

core-shell structured morphology. Namely, the

mesoporous silica shell was coated on the surface of

single UCNP core in one-in-one fashion. The size of

UCNP@mSiO2-PEG nanospheres is about 86 nm. The

N2 adsorption/desorption isotherm and pore-size

distribution (Fig. S1, Supporting Information) of

UCNP@mSiO2-PEG indicates the mesoporous nature

of the materials, which is suitable for the loading of

drug molecules. Upon excitation with 980 nm

near-infrared laser, the resultant emission bands at

521 nm, 542 nm, and 652 nm can be ascribed to

2H11/2→4I15/2, 4S3/2→4I15/2, and 4F9/2→4I15/2 transitions of

activator Er3+ ions, respectively (Fig. 1(d)) [38].

In the subsequent preparation for

electrospinning composite fibers, PCL-gelatin (PG)

was chosen to modulate the viscoelasticity of the

precursor for electrospinning because PCL and

gelatin (PG) are recognized as safe and

biodegradable by the US FDA (Food and Drug

Administration) and CE (Conformit Europe) [39-41].

Furthermore, PG fibrous scaffold has promising

candidates in the fields of drug delivery and tissue

engineering [42-44]. In our current study, DOX-loaded

UCNP@mSiO2-PEG was encapsulated into PG

composite fibers including the other antiphlogistic


12 Nano Res.

drug MC to form novel dual drugs carrier

MC/UCNPS/DOX. Fig. 2 has illustrated the shape of

the resultant MC/UCNPS/DOX composite fibers

(CFs). From Fig. 2(a), one can see that an enormous

amount of fibers can be clearly observed. The length

of fibers ranges from several tens to hundred

micrometers while the diameter is about 0.4-0.6 μm.

The TEM image (Fig. 2(b)) presents that the

DOX-UCNP@mSiO2-PEG spherical nanoparticles are

evenly distributed within the PG composite fibers.

Additionally, in order to carry out the contrast

experiments, the spinning pieces of pure drug DOX

CFs, pure materials UCNPS CFs, single drug-loaded

UCNPS/DOX CFs and dual drugs-loaded

MC/UCNPS/DOX CFs can be obtained under

appropriate electrospun conditions, respectively, as

shown in Fig. 2(c). From Fig. 2(c), it can be seen that

all kinds of the spinning pieces have flat surface and

uniform thickness.

3.2. DOX release properties of all kinds of CFs in

vitro

To examine the drug release properties of all kinds of

CFs, we comparatively have investigated their

release behaviors of an anti-cancer drug DOX.

Cumulative DOX release from

DOX-UCNP@mSiO2-PEG, DOX CFs, UCNPS/DOX

CFs and MC/UCNPS/DOX CFs at PBS buffer with

different pH is shown in Fig. 3. At pH 7.4, all samples

show similar release profile with lower DOX release

amount of only 7.5% (Fig. 3(a)). However, the DOX

release at pH 6.2 displays the fast rate in initial 4 h

and then a slow and continuous release in

DOX-UCNP@mSiO2-PEG, UCNPS/DOX CFs, and

MC/UCNPS/DOX CFs (Fig. 3(b)). This pH-responsive

DOX release profile can be explained as follows. The

lower pH value made surface zeta-potential of the

SiO2 layer more positive, which attenuated

electrostatic interaction between SiO2 and DOX

molecules with positive charges, leading to the faster

DOX release from carrier [45]. On the other hand, the

cumulative drug release behaviors for the

UCNPS/DOX CFs, MC/UCNPS/DOX CFs are


13 Nano Res.

different from those of DOX-UCNP@mSiO2-PEG and

DOX CFs. Additionally, from Fig. 3(b), the release

behaviors of DOX from DOX-UCNP@mSiO2-PEG

nanoparticles and DOX CFs both have presented a

burst release. The DOX cumulative release amount is

48% for DOX-UCNP@mSiO2-PEG nanoparticles and

37% for DOX CFs within 12 h, then the drug release

reaches to a plateau after 24 h. In contrast, the DOX

in the UCNPS/DOX and MC/UCNPS/DOX CFs has

shown a persistent and long-term release behavior,

with a cumulative release amount of DOX up to

about 20% within 12 h and 34% after 128 h.

Compared with the burst release of

DOX-UCNP@mSiO2-PEG and DOX CFs, the

sustained releasing can last for more than 120 h. The

reasons for the starting burst release in UCNPS/DOX

and MC/UCNPS/DOX CFs can come down to both

the free distribution of DOX outside of

UCNP@mSiO2-PEG and the swelling of PG in fiber

matrices. Then the afterward process of sustained

releasing can attributed to the fact that in the

presence of UCNP@mSiO2-PEG a great majority of

DOX was encapsulated in the mesopores of SiO2. In

this situation, the DOX release has to traverse the two

barriers of both mesoporous SiO2 and PG. So the

DOX release from UCNPS/DOX and

MC/UCNPS/DOX CFs presented a persistent and

long-range behavior at pH 6.2 with a sluggish

releasing after 128 h. To sum up, this ingenious

architecture design of dual-drugs delivery system

can solve effectively the problem of drug burst

release to some extent in our previous systems [30-32].

3.3. The releases properties of MC and DOX in

MC/UCNPS/DOX CFs

To find out whether the DOX-UCNP@mSiO2-PEG

influenced the release of another drug MC in CFs, the

MC releasing behaviors of only MC loaded CFs

(labeled as MC) and MC/UCNPS/DOX CFs were

measured. The result is displayed in Fig. 4. Owning

to the random distribution of MC in the fiber

matrices, the MC in both MC CFs and

MC/UCNPS/DOX CFs has displayed a burst release


14 Nano Res.

behavior in the starting 15 h. The cumulative release

amounts of MC after 64 h are about 62% and 57%,

respectively. The above outcomes suggest that

DOX-UCNP@mSiO2-PEG in the PG CFs has no

obvious effect on the release of MC. On the contrary,

the presence of MC also has little impact on the

release of DOX in the dual drugs delivery system

MC/UCNPS/DOX CFs comparing with single DOX

loaded UCNPS/DOX CFs (Fig. 3(b)). In other words,

the release behaviors of two drugs DOX and MC are

non-interfering. In this way, their respective

advantages can be very well displayed in tumor

therapy, which is very important for boosting the

sit-specific therapeutic efficacy and wound healing.

3.4. Up-conversion luminescence (UCL) and

magnetic resonance (MR) imaging effect of

MC/UCNPS/DOX CFs in vivo

The UCNP core in composite material endows it with

concurrent up-conversion luminescence and

magnetic properties, so we evaluate the application

of MC/UCNPS/DOX CFs in UCL/MRI dual modal

imaging in vivo. The MC/UCNPS/DOX CFs were

pasted on the surface of the tumor of the

tumor-bearing mice. The diffusion of nanoparticles

inside the tumor was monitored by the change of

upconversion luminescence in vivo. As shown in

Figure 5(b), the tumor site covered by

MC/UCNPS/DOX CFs has displayed red emission at

0 h, pink emission at 24 h and white-green emission

at 96 h under 980 nm laser excitation, respectively.

The above phenomenon can be explained as follows.

Since the UC emission bands in the green region

overlap with the broad absorbance of DOX centered

at about 480 nm, leading to occurrence of energy

transfer from UCNP to DOX. As such, the green

fluorescent of UCNPS in MC/UCNPS/DOX CFs was

quenched after loading DOX, and red emission was

obtained after the spinning piece was pasted on

tumor site. Subsequently, owing to the gradually

release of DOX from MC/UCNPS/DOX CFs with

extended time, the energy transfer from UCNP to

DOX has become weaker and weaker, which leads


15 Nano Res.

directly to the change of luminescence color (Figure

5(b)).Thus, the green emission becomes stronger,

which is quite consistent to the change of the

spectrum in Fig. S2. (Supporting Information). This

outcome suggests that MC/UCNPS/DOX CFs can act

as a UCL imaging agent in vivo to monitor DOX

releasing. From the relaxation rate (Fig. 5(c)) R1

(1.80304) (1/T1) versus different mass concentrations

of UCNPS in MC/UCNPS/DOX CFs pieces at room

temperature and the obvious lighting effect (Fig. 5(d))

in the tumor, it can be demonstrated that the

composite materials can be used as a T1-weighted MR

contrast agent in vivo because of the presence of

paramagnetic Gd3+ ions. This signal is probably

because some of UCNPS have entered tumor tissue

underneath the fiber-piece by diffusion mechanism.

The multimodal UCL/MR imaging combines the

advantages of enhanced sensitivity of luminescence

imaging, and high spatial resolution of MR imaging,

which is paramount for real-time monitoring the

evolution of disease [46, 47].

3.5. In vivo antitumor efficacy of CFs

In our current study, a series of experiments were

conducted in order to verify the capability of

inhibiting tumor of MC/UCNPS/DOX CFs. In this

experiment, the control group received no further

treatment and other four groups were treated with

DOX CFs, UCNPS CFs, UCNPS/DOX CFs and

MC/UCNPS/DOX CFs, respectively. In our

experiments, it is found that the heavier mice with

similar tumor volume will grow much healthier in

the same group. The mice in control group without

any treatment will become unhealthier and are

more likely to die than those of treatment groups. In

this case, the heavier mice will be selected as control

group to make sure that they are live until the end

of the experiment. During the entire experiments,

only a mouse in control group died and other mice

appeared lively without the signs of decreased

activity. Fig. 6 has illustrated the antitumor efficacy

of CFs in vivo. From the photograph of tumors of

each group in Fig. 6(a), one can see that the tumor


16 Nano Res.

sizes of MC/UCNPS/DOX group are smallest

among five groups. An even more important

finding is that the tumor of a mouse in

MC/UCNPS/DOX-treated group completely

disappeared at the end of experiment, as indicated

by the black circle. On the last day of experiment,

all of mice were executed. To assess the tumor sizes

and inhibition rates, the tumors were excised from

these mice and weighted. The mean tumor

inhibition (Fig. 6(b)) is about 96% for

MC/UCNPS/DOX CFs group and 95% for

UCNPS/DOX CFs group relative to the control

group, respectively, which is much higher than that

of pure DOX (61.8%) and our previous systems [34].

The enhanced tumor inhibition of the

MC/UCNPS/DOX CFs can be elucidated from two

aspects. On one hand, it is related to the sustained

DOX release from UCNP@mSiO2-PEG and as well

as the accumulation of DOX in the intratumor once

implanted administration. On the other hand, it

should be credited to the anti-inflammatory drug

MC in MC/UCNPS/DOX CFs which can suppress

the inflammatory efficacy of the wound after the

surgery. As can be seen from the representative

pictures of mice in Fig. 6(c), the wound in

MC/UCNPS/DOX group has been completely

healed while the wound for mice in other group has

become inflamed after the surgery. The weight of

all mice is growing stably with the time extension,

as shown in Fig. 6(d). However, the tumors in the

control, UCNPS and DOX groups keep growing

while MC/UCNPS/DOX and MC/UCNPS groups

show the dramatic inhibition of the tumor growth

(Fig. 6(e)).

Although UCNPS/DOX CFs and

MC/UCNPS/DOX CFs groups have displayed

comparable antitumor efficacy, the cure rate of the

wound is different after surgery procedures. The

wounds of 3 out of 5 mice are healed in

MC/UCNPS/DOX CFs group, however, the wound

of only 1 mouse is healed and the wounds of other 4

mice are suppurative and inflamed in UCNPS/DOX


17 Nano Res.

CFs group. The unique role of anti-inflammatory

effect of MC is presented in Fig. 7. The routine

blood test has displayed that MC in

MC/UCNPS/DOX CFs can inhibit a surge of white

blood cell count (WBC) in quantity compared with

the UCNPS/DOX CFs group (Fig. 7(a)). Moreover,

from Fig. 7(b), one can also find that the wound of

the mouse in MC/UCNPS/DOX CFs group is in

better condition in contrast to that in UCNPS/DOX

CFs group with redness and inflammation after 5

days of the wound suture surgery. The above

results show that the MC in MC/UCNPS/DOX CFs

has significant effect on inhibiting inflammation of

wound after surgery. Thus this kind of dual drugs

delivery system will be perfectly fit for local

diagnosis and treatment, especially for those

patients receiving complete tumor resection or

cytoreductive surgery.

3.6. Toxicity assessment of UCNPS CFs, DOX CFs,

UCNPS/DOX CFs and MC/UCNPS/DOX CFs

The analysis results of gross anatomy and

pathomorphology examinations have suggested that

all of the organs, including heart, spleen, liver, and

kidney are health with no visible inflammation,

necrosis or lesion after the treatment of UCNPS CFs,

DOX CFs, UCNPS/DOX CFs and MC/UCNPS/DOX

CFs. The above result indicates that the CFs as drug

carrier have excellent in vivo biocompatibility. We

also test potential toxic of the CFs on the treated mice

by biochemical and hematological analyses of blood.

The result is displayed in Fig. 8. Different organs

function signals indicate that the functions of the

liver, spleen, kidneys, and heart are quite normal.

This is possibly because the great majority of UCNPS

is concentrated underneath the fiber-piece. The

reason is probably that UCNPS is too big to traverse

the histocyte to circulatory system. So, the UCNPS

concentration in other organs is quite low and not

enough to induce adverse effects. This implies that

the composite fibers can be served as an implant

material for effective treatment of intratumors while

minimizing side effects.


18 Nano Res.

3.7. Biodistribution and release of drug in vivo

In order to research biodistribution of

MC/UCNPS/DOX CFs in vivo, the mice after pasting

MC/UCNPS/DOX fiber-pieces on the tumor were

sacrificed at different time intervals. Then the tumor

and major organs were collected and imaged by the

Maestro system. As shown in Fig. 9, ex vivo DOX

images of various organs have displayed high drug

accumulations in the tumor while weak signal is

observed in liver and kidney. Moreover, with the

extension of time, DOX fluorescence in the tumor

increases gradually and then decreases after 24 h. It is

because the great majority of DOX is concentrated

underneath the fiber-piece within the first 24 h, and

afterwards a part of DOX enters some organs

especially liver and kidney by blood circulation.

Since the DOX concentration in other organs is quite

low and not enough to induce adverse effects. These

findings indicate that the strategy for local

chemotherapy by implanting directly inside the solid

tumors provides alternative means for a safe,

efficient, and convenient chemotherapy. This style of

treatment can enhance the specificity of the drug

delivery, reduce the damage of the drugs to healthy

tissues and maximize the drug concentration at the

tumor site, leading to a greater inhibitory effect on

tumor growth.

3.8. Distribution of metabolic of

UCNP@mSiO2-PEG in MC/UCNPS/DOX CFs

To understand the biodistribution of

UCNP@mSiO2-PEG nanoparticles in CFs in vivo,

Kunming mice implanted with MC/UCNPS/DOX

were sacrificed at different time intervals. Then

tumors and principle organs were collected and

solubilized with HNO3 and H2O2 after the remaining

spinning pieces were removed by surgical operation.

From Gd3+ concentrations (Fig. 10) determined by

ICP-AES technique, it can be clearly seen that the

majority of UCNP@mSiO2-PEG nanoparticles are

concentrated at the tumor even after 7 days. The Gd3+

contents of organs nearly have kept unchanged. This

suggests that the UCNP nanoparticle embedded in


19 Nano Res.

MC/UCNPS/DOX CFs almost can’t enter the

circulatory system. This is different from the

behavior of DOX biodistribution in Fig. 9, in which a

part of DOX enters some organs especially liver and

kidney by blood circulation. This is probablely

because the small molecule DOX can be much easy to

escape from the electrospinning composite

fiber-pieces by slowly swelled and broken in ambient

conditions and enter blood circulatory system [48, 49].

But Gd3+ contained in UCNP@mSiO2-PEG

nanoparticles is possiblely too big to traverse the

histocyte to circulatory system. The above result has

proved that concentration of UCNP@mSiO2-PEG is

quite low in organs and not enough to induce

adverse effects.

4. Conclusion

Electrospun upconversion composite fibers dual

drugs delivery system was successfully assembled.

DOX-loaded

NaGdF4:Yb/Er@NaGdF4:Yb@mSiO2-polyethylene

glycol nanoparticles were incorporated into

antiphlogistic drug MC loaded poly(ε-caprolactone)

(PCL) and galatin to form MC/UCNPS/DOX CFs by

electrospinning. The resultant multifunctional

spinning pieces can be implanted directly to the

tumor site of mice by surgical procedures to fulfill

the orthotopic chemotherapy by the controlled

release of DOX from mesoporous SiO2 and the

upconversion fluorescence/magnetic resonance

dual model imaging through

NaGdF4:Yb/Er@NaGdF4:Yb embedded in

MC/UCNPS/DOX in vivo. What’s more, the MC in

MC/UCNPS/DOX CFs can suppress the

inflammatory responses, which helped to heal the

wounds in vivo. These results provide an

encouraging prospect of using drug loaded

electrospun nanofibers in orthotopic diagnosis and

treatment combined with presently employed

treatment protocols, especially for those patients

suffering from unresectable tumors or metastases

cancer.


20 Nano Res.

Acknowledgements

This project is financially supported by the National

Natural Science Foundation of China (NSFC

51332008, 51372243, 51422209), National Basic

Research Program of China ( 2014CB643803).

Supporting Information Available: N2

adsorption–desorption isotherms and mesopore size

distribution (the inset) of UCNP@mSiO2-PEG

nanocomposite (Fig. S1) UC emission spectra of

MC/UCNPS CFs and MC/UCNPS/DOX CFs under

980 nm laser excitation (Fig. S2). These material is

available free of charge via the Internet at

http://dx.doi.org.

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http://www.sciencedirect.com/science/article/pii/S1742706107000153

http://link.springer.com/search?facet-author=%22Sneh+Gautam%22

http://link.springer.com/search?facet-author=%22Sneh+Gautam%22

http://link.springer.com/search?facet-author=%22Amit+Kumar+Dinda%22

http://link.springer.com/search?facet-author=%22Amit+Kumar+Dinda%22

Scheme 1. The schematic diagram of the multifunctional MC/UCNPS/DOX electrospinning composite fibers as dual drugs delivery

system for synchronous UCL/MR imaging and therapy of tumor in vivo.


27 Nano Res.

Figure 1 TEM (a) and high-resolution (HR) TEM (b) images of NaGdF4:Yb/Er@NaGdF4:Yb (UCNP), TEM image of (c) and

up-conversion emission spectra (d) of UCNP@mSiO2-PEG as well as the corresponding digital luminescence photographs dispersed

in water under 980 nm laser excitation.


28 Nano Res.

Figure 2 TEM images (a, b) of MC/UCNPS/DOX CFs, as well as the digital photographs (c) of UCNPS, UCNPS/DOX,

MC/UCNPS/DOX and DOX spinning pieces.


29 Nano Res.

Figure 3 Cumulative DOX release from DOX-UCNP@mSiO2-PEG, DOX CFs, UCNPS/DOX CFs and MC/UCNPS/DOX CFs at pH

7.4 and pH 6.2 PBS buffer. Error bars were based on standard deviations (SD) of three times per group.


30 Nano Res.

Figure 4 Cumulative MC release from MC CFs and MC/UCNPS/DOX CFs at pH=6.2. Error bars were based on standard deviations

(SD) of three times per group.


31 Nano Res.

Figure 5 The schematic diagram (a) of in vivo up-conversion luminescence images and the corresponding luminescence colors (b) of

Kunming mice bearing tumors at different time point after in situ paste CFs patchs (60-100 mm2/mat, 8 pieces/mouse); relaxation rate

(c) R1 (1/T1) versus different mass concentrations of UCNPS in MC/UCNPS/DOX CFs pieces at room temperature using a 1.2 T

MRI scanner at different gadolinium concentrations; coronal MR images (d) of Kunming mice bearing tumors pre and post in situ

paste CFs patchs (60-100 mm2/mat, 8 pieces/mouse).


32 Nano Res.

Figure 6 The photographs (a) and tumor weight (b) of excised tumors from euthanized mice on the last day of experiment, as well as

the images (c) of representative Kunming mice with tumors, the body weight (d) and the tumor volume (e) recorded for mice after

treatment with UCNPS CFs, DOX CFs, UCNPS/DOX CFs, MC/UCNPS/DOX CFs and control group, Error bars were based on

standard deviations (SD) of four mice per group (n=4).


33 Nano Res.

Figure 7 White blood cell count (a) of MC/UCNPS/DOX CFs, UCNPS/DOX CFs and control groups as well as the photographs (b)

of wounds of MC/UCNPS/DOX CFs and UCNPS/DOX CFs 5 days after surgery. Error bars were based on standard deviations (SD)

of two mice per group (n=2).


34 Nano Res.

Figure 8 Systematic toxicity of composite fibers marerials on healthy Kunming mice: hematoxylin and eosin stained images (a) of

major organs and blood analysis data (b) of mice 17 days after treatment with UCNPS CFs, DOX CFs, UCNPS/DOX CFs,

MC/UCNPS/DOX CFs and control group, respectively. Error bars were based on standard deviations (SD) of four mice per group

(n=4).


35 Nano Res.

Figure 9 Biodistribution and in vivo drug (DOX) release of MC/UCNPS/DOX CFs. (a) Typical ex vivo images of the excised organs

examined by CRI Maestro 500 FL at 0.5 h, 2 h, 8 h, 12 h, 24 h, 2 d, 3 d, 5 d and 7 d after pasting MC/UCNPS/DOX fiber-pieces on

the tumor of Kunming mice. (b) Semi-quantitative fluorescence intensities of various organs determined at different time points.

Error bars were based on standard deviations (SD) of three mice per group (n=3).


36 Nano Res.

Figure 10 Biodistribution of UCNP@mSiO2-PEG in mice at different time points after treatment with MC/UCNPS/DOX CFs by

ICP-AES (concentration of Gd3+). Error bars were based on standard deviations (SD) of three mice per group (n=3).


Nano Res.

Electronic Supplementary Material



Yinyin Chen1,2, Shi Liu2, †, Zhiyao Hou1, Pingan Ma1, Dongmei Yang1,2, Chunxia Li, 1 () and Jun Lin,1()

Figure S1 N2 adsorption–desorption isotherms and mesopore size distribution (the inset) of UCNP@mSiO2-PEG nanocomposite.


Nano Res.

Figure S2. UC emission spectra of MC/UCNPS CFs and MC/UCNPS/DOX CFs under 980 nm laser excitation.

Address correspondence toJun Lin. [email protected]; Chunxia Li. [email protected]

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