CXCR4-Targeted and Redox Responsive Dextrin Nanogel for

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CXCR4-targeted and Redox Responsive Dextrin Nanogel for Metastatic Breast Cancer Therapy Feiran Zhang, Siman Gong, Jun Wu, Huipeng Li, David Oupicky, and Minjie Sun Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 26 Apr 2017 Downloaded from http://pubs.acs.org on April 27, 2017

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Biomacromolecules

CXCR4-targeted and Redox Responsive Dextrin Nanogel for Metastatic Breast Cancer Therapy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Feiran Zhang†, Siman Gong†, Jun Wu§, Huipeng Li†, David Oupicky*†‡, and Minjie Sun*† †

State Key Laboratory of Natural Medicines and Department of Pharmaceutics, China Pharmaceutical University, Nanjing, 210009, China, ‡Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA, and §Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University, Guangzhou, China

* Address for Correspondence: Minjie Sun, Professor State Key Laboratory of Natural Medicines Department of Pharmaceutics China Pharmaceutical University Nanjing, China 210009 Phone /Fax: +86 25 83271098 Email: [email protected] David Oupicky, Professor Center for Drug Delivery and Nanomedicine Department of Pharmaceutical Sciences University of Nebraska Medical Center Email: [email protected]

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ACS Paragon Plus Environment

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Abstract: The unsatisfied results of cancer therapy are caused by many issues and metastasis of

3

cancer cells is one of the major challenge.

It has been reported that inhibiting the

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SDF1/CXCR4 interaction can significantly reduce the metastasis of breast cancer cells to

5

regional lymph nodes and lung. Herein, a nanogel system equipped with the FDA-approved

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CXCR4 antagonist AMD3100 was developed and evaluated for its combined anti-metastatic

7

and tumor targeting effects. Briefly, a bioreducible cross-linked dextrin nanogel (DNG)

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coated with AMD3100 was designed to possess multiple functions, including CXCR4

9

chemokine targeting, inhibition of tumor metastasis and reduction-responsive intracellular

10

release of doxorubicin (DOX) to reduce the cells proliferation. The in vitro results confirmed

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that the DOX-loaded AMD3100-coated dextrin nanogel (DOX-AMD-DNG) was more

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effectively taken up by 4T1 breast cancer cells than DOX-DNG, and was significantly more

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cytotoxic to 4T1 cells than DOX-DNG. In biodistribution studies, the stronger fluorescence

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intensity of Cy7-AMD-DNG than Cy7-DNG further confirmed that AMD3100 mediated

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tumor targeting in vivo. AMD3100-coated DOX-DNG also exhibited a distinct anti-metastatic

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effect and CXCR4 antagonistic activity by inhibiting CXCR4-mediated cell invasion in 4T1

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and U2OS cells. Moreover, DOX-AMD-DNG displayed superior anti-cancer activity and

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anti-metastatic effects in orthotopic breast cancer-bearing Balb/C mice. In summary, the

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multi-functional DOX-AMD-DNG can effectively target the tumor site and dually impede

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cancer progression and metastasis.

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Keywords：：

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Dextrin nanogel; CXCR4 targeting; metastatic breast cancer; redox sensitive nanogel; anti-

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metastasis

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1. Introduction

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Breast cancer is one of the most common malignant diseases in women. The majority of

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deaths of breast cancer is caused by the tumor metastasis, which frequently follows a specific

5

pattern of dissemination.1, 2 Currently, there is still no effective modality in clinical use for

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advanced breast cancer in which metastasis has already occurred at the late stage of the

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cancer.3 It has been proposed that the chemokine receptor CXCR4 and its cognate ligand

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CXCL12 (SDF-1) regulate directional invasion of breast cancer cells to certain sites of

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metastasis, such as lymph nodes, lung, liver and bone marrow, which express high levels of

10

SDF-1.4,

5

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significantly inhibit the metastasis of breast cancer cells to regional lymph nodes and lung.

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There is growing evidence that an FDA-approved CXCR4 antagonist, Plerixafor (AMD3100),

13

inhibits tumor growth, induces tumor apoptosis and prevents metastatic spread. Since CXCR4

14

is overexpressed on the surface of the cell membrane,6, 7 it is feasible to combine a CXCR4

15

antagonist to provide both anti-metastasis and tumor targeting functions for enhanced cancer

16

therapy.

The neutralizing the interaction of SDF-1 with its receptor CXCR4 can

17

Nanogels are hydrophilic nano-sized hydrogels created by physical or chemical

18

crosslinking. Nanogels are widely used for their excellent drug loading capacity, sensitive

19

response to stimuli in the external environment, and superior stability.8 Nobuyuki M et al.

20

developed a new acid-labile CHP (acL-CHP) nanogel that remained stable under normal

21

physiological pH conditions but was degraded to release its cargo under acidic conditions.

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The nanogel was an effective nanocarrier in vivo for delivering protein for bone formation and

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for vaccines targeted to tumors and mucosal tissue.9 Herein, in this study, a dextrin (Dex)

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nanogel platform was developed for targeting CXCR4 for treating of metastasis of breast

25

cancer. Dex nanogels are non-cytotoxic and effective carriers for anti-tumor drug delivery 3



1

with high stability in the blood circulation and controlled drug release in tumor tissue.10 In

2

addition, Dex contains numerous hydroxyl groups which can easily be modified to provide

3

stimulus-responsive properties for controlled drug release.11 Redox-responsive delivery

4

system tend to undergo rapid cleavage in the reducing intracellular environment, thus

5

achieving burst release. It was proved to be an efficient strategy for enhanced drug delivery.12,

6

13

7

We hypothesized that AMD3100-equipped redox sensitive nanocarriers can combine the

8

ability to target CXCR4-overexpressing tumors, reduce metastasis and inhibit tumor growth

9

into one cancer therapy (Scheme 1). We investigated the cellular uptake and cytotoxicity of

10

the AMD3100-modified DOX-encapsulating Dex nanogel (DOX-AMD-DNG) in the murine

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breast cancer 4T1 cell lines. We also present the result of studies showing that AMD3100

12

antagonized CXCR4 in U2OS cells, inhibited cell invasion through Matrigel, and mediated

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tumor targeting in vivo. Finally, we demonstrated the anti-cancer and anti-metastasis efficacy

14

of DOX-AMD-DNG in breast cancer-bearing Balb/C mice.

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2. Materials and Methods

3

2.1 Materials

4

Dex was kindly donated by Roquette Corporate. Doxorubicin (DOX, content > 98%) was

5

purchased from Nanjing Chemlin Chemical Industry Company. AMD3100 was purchased

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from Biochempartner. Cystamine dihydrochloride and DL-dithiothreitol (DTT) were

7

purchased from Shanghai DiBo Company. Glutathione (GSH), 1-(3-dimethylaminopropyl)-3-

8

ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased

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and used as received from Aladdin. Cy7 NHS ester (Cy7-se) was purchased from Nanjing

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Well-Offer Biotechnology Co. Thiazolyl blue tetrazolium bromide (MTT) was obtained from

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Amresco (Solon, Ohio, USA). Human SDF-1 was from Shenandoah Biotechnology, Inc.

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(Warwick, PA). Trypsin, penicillin, streptomycin, fetal bovine serum (FBS) and Roswell Park 5



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Memorial Institute 1640 cell culture medium (RPIM-1640) were from Hyclone (Waltham,

2

USA). All other experimental chemicals were of reagent grade.

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2.2 Cell culture and animals

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Murine breast cancer 4T1 cells and human epithelial osteosarcoma U2SO cells which

5

stably express the human CXCR4 receptor fused to the N-terminus of enhanced green

6

fluorescent protein (EGFP) were employed. The 4T1 cells were cultured in RPMI 1640

7

medium supplemented with 10% FBS and 1% Pen-Strep. The U2OS cells were cultured in

8

DMEM supplemented with 2 mM L-glutamine, 10% FBS, 1% Pen-Strep and 0.5 mg/ml

9

G418. All the cells were maintained at 37 °C in an incubator with 5% CO2 and 90% relative

10

humidity. The cells were sub-cultured approximately every other day at 80% confluence

11

using trypsin (0.25%, w: v) at a split ratio of 1:3.

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Balb/C mice (female, weight 20 ± 2 g, n = 50) were purchased from China

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Pharmaceutical University animal center. All the animal experiments were performed in

14

compliance with the National Institutes of Health Guide for the Care and Use of Laboratory

15

Animals, China (2005).

16

2.3 Synthesis of Dex-COOH

17

Carboxylated Dex (Dex-COOH) was first synthesized by dissolving Dex in a mixture of

18

DMSO and DMF at a ratio of 1:1 (v: v) at 50 °C. 4-Dimethylaminopyridine (DMAP) and

19

succinic anhydride (SA) were further added and the mixture was stirred under a protective

20

nitrogen atmosphere for 24 h. To remove the excess reactants, the mixture was precipitated

21

with ice-cold ethyl ether (v: v=10:1). The solution was centrifuged at 5000 rpm for 5 min and

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the supernatant was discarded. Dex-COOH was purified by dialysis (MWCO 8000-12000)

23

and freeze-dried.

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2.4 Synthesis of Dex-SH 6


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Thiolated Dex (Dex-SH) was obtained by reducing the disulfide bond of cystamine-

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modified Dex (Dex-cys). Dex-COOH was first dissolved in a mixture of DMSO and DMF (v:

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v = 1:1) and the solution was stirred under a protective nitrogen atmosphere. EDC and NHS at

4

a molar ratio of 1:1.5 were further added to the solution with continued stirring for 1 h.

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Cystamine dihydrochloride was added drop-wise and mixed for another 12 h. The reaction

6

mixture was precipitated with ice-cold ethyl ether and Dex-cys was obtained by dialyzing

7

(MWCO 8000-12000) and freeze-drying.14 Then, Dex-cys was treated with a 6 molar excess

8

of DTT at pH 8.5 and stirred for 4 h. The reaction solution was then adjusted to pH 3.5 with 1

9

M HCl and dialyzed for 6 h. The final product was obtained by freeze-drying.

10

2.5 Characterization of Dex derivatives

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The 1H-NMR spectra of Dex, Dex-COOH and Dex-cys were measured on a Bruker

12

AVANCE 500 spectrometer (Bruker, Switzerland). The infrared (IR) spectra were collected

13

by a Nicolet Impact 410 spectrometer (Thermo Company, USA) within the range 400-4000

14

cm-1.

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2.6 Preparation of DNG, DOX-DNG and DOX-AMD-DNG

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Dex nanogel (DNG) was prepared by a suspension method. 5 mg Dex-SH was dissolved

17

in 1 ml pH 8.0 Tris buffer solution and vortexed for 1 min. Dex nanogel with encapsulated

18

DOX (DOX-DNG) was further prepared by adding 5 mg/ml DOX with stirring, then standing

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for 1 h. The solution of DOX-DNG was subjected to ultrafiltration (Eppendorf AG 22331

20

Hamburg, MWCO 5000) at 5000 rpm for 20 min to remove free DOX and then dispersed in 1

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ml of 2 mg/ml AMD3100 solution to prepare AMD3100-coated DOX-DNG (DOX-AMD-

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DNG).

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2.7 Characterization of DNG, DOX-DNG and DOX-AMD-DNG

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2.7.1 Particle size and zeta potential characterization 7



1

The particle size and zeta potential of DNG, DOX-DNG and DOX-AMD-DNG were

2

determined with a Zetasizer Nano ZS laser particle size analyzer (Malvern, UK).

3

2.7.2 Encapsulation efficiency and drug loading content

4

The free DOX was separated from the DOX-DNG using the centrifugation method as

5

described in 2.6. The sample was demulsified with methanol and the solution in the bottom

6

layer was collected. The concentration of DOX was measured with HPLC. The drug loading

7

content (DL %) and encapsulation efficiency (EE %) of DOX-DNG were calculated as

8

follows:

9 10 11

DOX was measured by HPLC (equipped with a LC-20A Shimadzu pump and a SPD-10A

12

Shimadzu UV detector) on a Dikma Diamonsil C18 (5 µm, 150 mm × 4.6 mm) column eluted

13

with a mobile phase consisting of a 55:45 (v/v) mixture of acetonitrile and 0.2% acetic acid-

14

water at a flow rate of 1 ml/min at 30 °C.

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2.8 In vitro drug release

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The in vitro GSH-induced DOX release from the nanogel was analyzed by the dialysis

17

method.15 1 ml DOX-AMD-DNG at a DOX concentration of 700 µg/ml or free DOX at 700

18

µg/ml was placed into a tightly sealed dialysis bag (MWCO 8000-12000). Subsequently, the

19

dialysis bags were immersed into 20 ml PBS (pH 7.4 with 10 µM GSH or pH 5.0 with 10 mM

20

GSH) and put in 37 °C water baths with mechanical shaking at 100 rpm. At each

21

predetermined time interval of 0, 0.5, 1, 2, 4, 7, 12 and 23 h, 1 ml of dissolution medium was

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withdrawn from each vial and replaced with the same amount of pre-warmed fresh medium.

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The amount of released DOX in the withdrawn samples was determined with a fluorescence

24

spectrophotometer using a λex of 480 nm to determine the concentration of dissociated DOX 8


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and thus to calculate the cumulative release rate. The DOX release profile from the DOX-

2

DNG in the presence of 10 µM GSH (pH 7.4) was studied as the extracellular negative

3

control.

4

2.9 In vitro cytotoxicity study

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The cytotoxicity of DOX-AMD-DNG was evaluated by MTT assay.16 4T1 cells were

6

seeded in 96-well plates at a density of 1×104 cells/well. After 24 h of culture, the culture

7

medium was removed and the cells were incubated with 200 µl of DOX, DOX-DNG or DOX-

8

AMD-DNG at concentrations of 0, 0.25, 0.5, 1, 2, 4, 8 and 16 µg/ml for 24 h. Then, 20 µl of

9

MTT solution (5 mg/ml) was added to each well and incubated for an additional 4 h. After

10

that, the medium was removed and 150 µl of dimethyl sulfoxide was added to dissolve the

11

crystals formed by living cells. Absorbance at 570 nm was measured using a microplate

12

reader. Cell viability was expressed as a percentage of the absorbance to that of the control

13

experiment without treatment. At the same time, the cytotoxicity of DNG to 4T1 cells was

14

also determined by MTT assay as described above.

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2.10 Uptake of DOX-AMD-DNG by 4T1 cells

16

In order to verify the CXCR4 receptor targeting property while sensitively visualizing

17

the intracellular behavior of the nanogels, the cellular uptake of DOX-loaded AMD-DNG was

18

investigated by combining flow cytometry and confocal laser scanning microscope (CLSM).17

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For CLSM observation, 4T1 cells (1×104 cells/well) were seeded into glass-bottom

20

culture dishes for 24 h. When the cells reached 60-70% confluence, the culture medium was

21

replaced with DOX, DOX-DNG or DOX-AMD-DNG containing DOX at a concentration of 1

22

µg/ml. The treated cells were cultured at 37 °C for 4 h, then washed three times with PBS to

23

stop cellular uptake and fixed with 4% paraformaldehyde for 15 min. After the cell nuclei

24

were stained with DAPI for 15 minutes, the cellular uptake of NLCs was visualized with a

25

CLSM (Zeiss, Germany). 9



1

For flow cytometer (FCM) analyses, 4T1 cells (5 × 104 cells/well) were seeded into 24-

2

well plates and incubated in RPMI 1640 medium for 24 h. The culture medium was then

3

replaced with DOX-AMD-DNG containing DOX at a concentration of 0.25, 0.5 or 1 mg/ml

4

and the cells were further cultured at 37 °C for 4 h. The untreated cells were used as a

5

negative control. The cells were rinsed with cold PBS three times to stop uptake. The cells

6

were collected by trypsinization and the fluorescence intensity was measured using a flow

7

cytometer (BD FACS Calibur, USA). To evaluate time-dependent cellular uptake behavior,

8

4T1 cells were also incubated with DOX-AMD-DNG containing DOX at a concentration of

9

0.5 mg/ml at 37 °C for 1, 2 or 4 h. Cellular uptake procedures were performed as described

10

above.

11

To investigate the uptake of DOX-AMD-DNG mediated by CXCR4 targeting, 4T1 cells

12

were incubated with free DOX, DOX-DNG and DOX-AMD-DNG containing DOX at a

13

concentration of 0.25, 0.5 or 1.0 µg/ml for 4 h. A fourth group of cells was pre-incubated with

14

free AMD3100 of 0.6 µg/ml for 1 h before treatment with DOX-AMD-DNG at a

15

concentration of 0.25, 0.5 or 1.0 µg/ml for 4 h. 4T1 cells were also incubated with the above

16

formulations containing DOX at a concentration of 0.5 mg/ml at 37 °C for 1, 2 or 4 h. The

17

cells were collected by trypsinization after rinsing with cold PBS three times and the

18

fluorescence intensity was measured using a flow cytometer.

19

2.11 CXCR4 antagonistic activity

20

CXCR4 antagonism of DOX-AMD-DNG was determined by CXCR4 redistribution

21

assay. U2OS cells expressing functional EGFP-CXCR4 fusion protein were seeded at a

22

density of 8000 cells per well in black 96-well plates with optical bottoms 24 h. The cells

23

were first washed twice with 100 µl assay buffer (DMEM supplemented with 2 mM L-

24

glutamine, 1% FBS, 1% Pen-Strep and 10 mM HEPES) and then incubated with AMD3100,

25

DNG and AMD-DNG in assay buffer containing 0.25% DMSO at 37 °C for 30 min and 10 10


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Biomacromolecules

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nM SDF-1 was added for 1 h.18 AMD3100 (0.2 µg/ml) was used as the positive control. Cells

2

treated with SDF-1 alone were used as the negative control. After 1 h incubation at 37 °C, the

3

cells were fixed with 4% formaldehyde at room temperature for 20 min and washed 4 times

4

with PBS. The cell nuclei were stained with 0.2% crystal violet in PBS. Images were taken

5

with an EVOS fluorescence microscope at 20×.

6

2.12 Cell invasion assay

7

The upper sides of the transwell inserts were coated with 40 µl Matrigel diluted 1:3 (v/v)

8

with serum-free medium. The 24-well plates with coated inserts were then placed in a 37 °C

9

incubator for 2 h. 4T1 cells were trypsinized and resuspended with AMD3100, DNG or

10

AMD-DNG in serum-free medium for 30 min, then added to the inserts at a final

11

concentration of 1 × 104 cells in 300 µl medium per insert. 20 nM SDF-1 in serum-free

12

medium as the chemo-attractant was then added to the corresponding wells in the companion

13

plate. After 24 h, cotton swabs were used to remove the non-invaded cells on the upper

14

surface of the insert membrane. The invading cells were then fixed and stained by dipping the

15

inserts into crystal violet solution for 10 min.18 The images were taken with an EVOS xl

16

microscope. Five 40 × imaging areas were randomly selected for each insert and each sample

17

was performed in triplicate.

18

2.13 In vivo tumor accumulation of Cy7-AMD-DNG

19

For in vivo distribution studies, we used a fluorescent probe, Cy7-se, to replace DOX in

20

the nanogel. The Cy7-se-loaded nanogel was prepared using the procedures described in

21

Section 2.6. The biodistribution of Cy7-AMD-DNG was investigated in a syngeneic mouse

22

tumor model using the 4T1 cell line in female Balb/C mice. Balb/C mice were anesthetized

23

and then each animal was injected with 5 × 106 4T1 cells in 100 µl PBS into the mammary fat

24

pad. Solid tumors were allowed to form over a period to reach a volume ranging from 50 to 11



1

100 mm3. Cy7-DNG and Cy7-AMD-DNG were intravenously injected at a dose of 200 µg/kg

2

(Cy7/body weight) and AMD3100 was injected at 2 mg/kg. At each predetermined time, the

3

tumor-bearing mice were anesthetized and imaged with an in vivo imaging system (Image

4

Station In-Vivo FX, Kodak, USA). The excitation wavelength of the in vivo imaging was set

5

to 720 nm to excite the Cy7.

6

2.14 Anti-metastatic and anti-tumor activity in vivo

7

The syngeneic breast tumor model was established as described in Section 2.13. Solid

8

tumors were allowed to form over a period of 1-2 weeks to reach a volume ranging from 50 to

9

100 mm3.19 For treatments, tumor bearing mice were randomly allocated to 5 groups (n=5)

10

and treated with the following formulations via the tail vein: (1) saline, (2) free DOX, (3) free

11

AMD3100, (4) DOX-DNG, (5) DOX-AMD-DNG. The formulations were injected every

12

other day (5 times in total) from day 0 to day 8. Each of the injected doses was normalized to

13

5 mg/kg DOX and 2 mg/kg AMD3100. The tumor volumes and the mouse body weights were

14

measured every other day. The tumor volumes were calculated by the following formula:

15

tumor volume V (mm3) = 1/2 × length (mm) × width (mm2). At day 21, all of the animals

16

were euthanized and sacrificed, and the tumors were excised and weighted. The lungs were

17

excised and fixed in 10% formalin solution, then embedded in paraffin for histological

18

hematoxylin and eosin (H & E) staining. Representative pictures were taken of the lungs and

19

tumors and of the corresponding H & E-stained sections.

20

2.15 Statistical analysis

21

All the results are expressed as mean ± standard deviation (SD) unless otherwise noted.

22

When appropriate, data were compared by Student’s t-test with P ≤ 0.05 as the minimal level

23

of significance

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3. Results and discussion

2

3.1 Characterization of Dex derivatives

3

Dex is a natural polysaccharide containing D-glucose units linked by α-(1→4) glycosidic

4

bonds, has high biocompatibility and degradability.20 Many studies have proved its clinical

5

tolerability and high level of absorption, leading to its wide use as a biodegradable material

6

for targeted delivery of therapeutics.21 In the present study, the thiolated Dex (Dex-SH) was

7

first synthesized by reacting the carboxyl group of Dex-COOH with the amine group in

8

cystamine, as shown in Scheme 1. The Dex-SH was synthesized in three steps. First, Dex was

9

reacted with a mixture of DAMP and SA to obtain Dex-COOH with a yield almost 90%.

10

Then cystamine was grafted to the Dex-COOH to obtain Dex-cys in a DMF/DMSO mixture

11

at room temperature. DTT was further added to the solution of Dex-cys to break the disulfide

12

bonds to obtain the Dex-SH. It was necessary to perform the dialysis at acid pH to prevent the

13

cross-linking of the thiol groups.

14

The 1H-NMR spectra of Dex, Dex-COOH and Dex-cys are shown in Figure 1. 1H NMR

15

spectrum of dextrin (300 MHz, D2O, ppm) demonstrated the peaks between δ = 3.376-3.950,

16

5.358 owing to the H2-H6 protons and H1 proton. The 1H-NMR spectrum of Dex-COOH in

17

Figure 1B shows a new proton signal at δ2.7 ppm due to the introduced SA methylene

18

protons. The signals at δ1.3 ppm corresponds to the protons on the amino group of cystamine.

19

Based on the NMR peak areas, we calculated the degree of substitution (DS) of carboxyl and

20

cystamine as 49.64% and 13.1%, respectively. The IR spectra of the Dex derivatives are

21

shown in Figure S1. The spectrum of Dex-COOH was similar to that of Dex except for

22

strong absorption bands at 1567 and 1730 cm-1. These bands were assigned to the introduction

23

of a carbonyl group (C=O) and methylene group (-CH2-). The spectrum of Dex-cys exhibited

24

a decrease in the absorption band at 3436 cm-1 due to replacement of the hydrogen atom on

25

the carboxyl group. 13



1 2

The thiols were quantified via Ellman’s assay,22 which reflected the presence of the

3

absorption band of DTNB2− at 412 nm. Our studies revealed that the degree of thiol

4

functionalization was 12.45%.

5

3.2 Preparation and characterization of Dex nanogel

6

Dex nanogel (DNG) was easily constructed in aqueous medium by self-crosslinking at

7

room temperature after vortexing. The dextrin derivatives contain units with numerous thiol

8

groups that allow the formation of disulfide bridges among dextrin backbones. These covalent

9

bonds are the main interactions to form the 3D network. Doxorubicin, as a model

10

chemotherapy drug, was mixed with the aqueous solution of Dex-SH to form DOX-DNG.

11

The encapsulation efficiency (EE) and drug loading efficiency (DL) of DOX-DNG were

12

optimized with different ratios of DOX and Dex (w/w), which are shown in Figure S2 (A). 14


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Biomacromolecules

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The optimum ratio of DOX to Dex was 1:2.5, which gives an EE of 38.04 % and a DL of

2

13.45 %. The high efficacy indicated that the cross-linking of the DNG and encapsulation of

3

DOX within the cross-linked DNG was more successful than in other Dex delivery systems.21

4

To create multifunctional DNG, the commercially available CXCR4 antagonist

5

AMD3100 was added to DOX-DNG solution to form DOX-AMD-DNG by electrostatic

6

interaction.23 The zeta potentials of DNG, DOX-DNG and DOX-AMD-DNG are shown in

7

Figure S2 (B). After adsorption of AMD3100, the zeta potential of DNG changed from

8

negative to a positive charge of 5 mV, confirming the effective coating by amine-rich

9

AMD3100.18

10

Meanwhile, the disulfide bonds are predicted to mediate the redox-responsiveness of the

11

nanogel, so that the nanogel will disintegrate in response to a high concentration of

12

intracellular glutathione (GSH) while remaining stable in the blood circulation. Transmission

13

electron microscopy (TEM) was used to visualize the diameter of nanogel. As characterized

14

by TEM, DOX-AMD-DNG existed as spherical nanostructures with an average diameter of

15

about 150 nm (Figure 2A). However, as shown in Figure 2B, after incubation with GSH, the

16

particle size increased sharply to over 400 nm and DOX-AMD-DNG appeared as irregular

17

spheres resulting from the breakage of the disulfide bonds. The size measured by TEM was

18

smaller than that determined by DLS, probably due to the dehydration during TEM sample

19

preparation, while the DLS measurements were more sensitive to the larger nanogel.24 In

20

addition, the dark staining in the TEM images indicated that the tungstophosphoric acid,

21

which is used to stain the samples, has penetrated deep into the DNG. These results revealed

22

that the network of DOX-AMD-DNG was destroyed with the high GSH concentration. This

23

will help the nanogel to disassemble in the cytoplasm and release the DOX into the nucleus.

15



1 2

The stability of DOX-AMD-DNG was investigated by incubation in PBS buffer (pH =

3

7.4 and pH = 5.0) and mouse plasma for 48 h at room temperature and measuring the particle

4

size at various time-points by DLS. Figure S3 revealed that DOX-AMD-DNG was rather

5

stable in pH 7.4 PBS and pH 5.0 PBS, as well as in plasma, which mimics the extracellular

6

environment. The sizes of DNG were smaller than those in PBS mainly due to the complex

7

compositions in plasma.

8

3.3 In vitro reduction-triggered drug release

9

The cross-linked DNG was expected to effectively protect DOX under normal

10

physiological conditions and to release the DOX in acidic and reducing intracellular

11

environments. The release behavior of DOX from DOX-AMD-DNG was investigated by

12

dialysis in various PBS solutions. Several features can be observed in the DOX release

13

profiles (Figure 3). Almost 90% of free DOX was released in 12 h, while only 29.2% of the

14

drug was released from DOX-AMD-DNG under non-reducing conditions (pH 7.4 and pH 5.0,

15

10 µM GSH). The results revealed that DNG prevented the release of DOX in conditions that

16

mimic the normal physiological environment. This can be ascribed to the impact of the cross-

17

linked structure. In PBS pH 5.0 and in the presence of 10 mM GSH, 70.3% of the drug was

18

released from DOX-AMD-DNG at 12 h. It should be noted that DOX-AMD-DNG had a 16


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similar release profile to free DOX under the reducing conditions, indicating that the redox-

2

sensitive disulfide bond should have a profound effect on intracellular DOX release. The in

3

vitro release studies confirmed that DOX release from DOX-AMD-DNG was triggered by

4

GSH. Thus, the prepared DNG are expected to rapidly release the encapsulated DOX in the

5

presence of high GSH concentrations in cancer cells.

6 7

3.4 In vitro cytotoxicity studies

8

Biocompatibility of DOX-AMD-DNG was evaluated by incubating it at different

9

concentrations with 4T1 murine breast tumor cells. We first incubated cells with Dex alone as

10

a control. The MTT assay results in Figure 4A show that Dex was highly biocompatibility at

11

concentrations ranging from 2.5 to 500 µg/ml after 24 h of treatment. The results for free

12

DOX, DOX-DNG and DOX-AMD-DNG are shown in Figure 4B. Compared with the free

13

DOX, the viability of 4T1 cells was slightly reduced by incubation with DOX-DNG. In

14

addition, DOX-AMD-DNG showed a much stronger inhibition of the growth of 4T1 cells.

15

We ascribe this difference to the AMD3100 on the surface of the nanogel, which is predicted

16

to accelerate the internalization of the DOX-AMD-DNG into the cancer cells in part by

17

interacting with CXCR4 and in part by non-specific uptake due to increased zeta potential of

18

DOX-AMD-DNG.25 These results further confirmed potential benefits of active CXCR4

19

targeting to achieve high cytotoxicity of DOX-AMD-DNG. 17



1 2

3.5 Intracellular distribution of DOX-AMD-DNG

3

Next, to investigate the intracellular distribution behavior of free DOX, DOX-DNG and

4

DOX-AMD-DNG, we carried out confocal microscopy and flow cytometry studies on 4T1

5

cells.

6

As shown in Figure 5, after incubation with equal concentration of DOX, cells treated

7

with DOX-DNG for 4 h exhibited a stronger fluorescence intensity than cells incubated with

8

free DOX, indicating that the nanogels mediated faster intracellular DOX uptake. The nuclei

9

of DOX-AMD-DNG treated cells were more fluorescent, which revealed that the increased

10

uptake of DOX-AMD-DNG facilitated higher nuclear delivery of DOX than incubation with

11

free DOX and DOX-DNG. The above results indicated that DOX-AMD-DNG is efficiently

12

taken up by CXCR4-overexpressing 4T1 cells and efficiently releases DOX and delivers it

13

into the nuclei of the cells.

18


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1 2

Flow cytometry showed the cellular uptake of DOX-AMD-DNG was about 2.6-fold and

3

1.8-fold greater than that of free DOX and DOX-DNG after 2 h incubation (Figure 6B). This

4

agrees well with the above confocal observations showing that the enhanced uptake of

5

nanogels is affected by the nanogel structure and AMD3100-mediated CXCR4 targeting. To

6

confirm involvement of the CXCR4 receptor in the cellular uptake of DOX-AMD-DNG, a

7

ligand competition experiment was performed in 4T1 cell line.26,

8

pretreated with a high dose of free AMD3100 to block CXCR4 receptors prior to DOX-

9

AMD-DNG treatment, the uptake of DOX-AMD-DNG sharply decreased (Figure 6A-B,

10

Figure S4). Given that 4T1 cells express high levels of CXCR4, our results suggest that

11

AMD3100 is responsible for the observed cell uptake behavior.

19


27

When the cells were


1 2

3.6 CXCR4 antagonistic activity

3

We evaluated the effect of inhibiting CXCR4 internalization into U2OS cells which

4

stably express EGFP-tagged CXCR4. We pre-treated the cells with AMD3100 or AMD-

5

DNG, then incubated the cells with SDF-1 before observing receptor internalization. The cells

6

incubated in SDF-1 alone displayed punctate fluorescence indicative of EGFP-CXCR4

7

internalization into endosomes (Figure 7A). In contrast, the cells incubated with SDF-1 after

8

AMD3100 pretreatment displayed weak fluorescence, which demonstrated that the CXCR4

9

antagonist AMD3100 inhibited CXCR4 internalization (Figure 7B). Cells pretreated with

10

AMD-DNG also showed strong CXCR4 antagonism (Figure 7D) when incubated with SDF-

11

1. The antagonism of CXCR4 by AMD-DNG was fully comparable to that of AMD3100. The

12

negative control (untreated) displayed the weakest fluorescence. The redistribution

13

experiment suggested that AMD-DNG inhibits the internalization of CXCR4 as effectively as

14

free AMD3100. We therefore predict that the AMD-DNG will have an anti-metastatic effect

15

in cells over-expressing CXCR4.

20


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1 2

3.7 Cell invasion assay

3

As discussed above, the CXCR4/SDF-1 axis is involved in migration of multiple types of

4

cancer cells, and CXCR4 antagonists are known to inhibit invasion of those cancer cells.

5

Binding of SDF-1 to the CXCR4 receptor on the cell membrane triggers activation of various

6

signaling pathways, leading to secretion of multiple MMPs that result in cancer cell invasion

7

and metastasis. Here we determined if antagonism of CXCR4 by the nanogel also led to

8

inhibition of cancer cell invasion. We used a Boyden chamber method to evaluate the SDF-1-

9

induced invasion of 4T1 cells. As shown in Figure 8, at an AMD3100 concentration of 0.5

10

µg/ml, AMD-DNG showed effective inhibition of cell invasion (60%). For comparison, the

11

inhibition of cell invasion achieved with the positive control (AMD3100) was 70%. The

12

negative control (DNG) did not show any significant inhibitory effect.

21



1 2

3.8 In vivo tumor accumulation of Cy7-AMD-DNG

3

The selectivity of in vivo tumor accumulation of anti-cancer drugs, delivered by the

4

nanogel, directly affects their therapeutic effects and possible side reactions. To demonstrate

5

the biodistribution behavior of the nanogel, we used a fluorescent probe, Cy7, to replace DOX

6

during preparation of the nanogel. Cy7-DNG and Cy7-AMD-DNG were intravenously

7

injected into 4T1-bearing Balb/C mice to track the in vivo targeting effect. As shown in

8

Figure 9, mice treated with Cy7-AMD-DNG exhibited much stronger fluorescent signals and

9

more obvious accumulation of Cy7 at the tumor site than those treated with Cy7-DNG. At 2

10

h, the fluorescent signals in the two groups were nearly the same intensity. However, the

11

tumor fluorescence in mice treated with Cy7-AMD-DNG became much stronger than that of

12

Cy7-DNG at 8 h and still maintained a high intensity at 24 h. Thus, the Cy7-AMD-DNG

13

clearly had a much better tumor targeting effect than the Cy7-DNG. These data, combined

14

with the cellular uptake studies in the previous section, indicated that Cy7-AMD-DNG

15

exhibited superior tumor targeting capacity, possibly due to the specific combination of the

16

AMD3100/CXCR4 interaction in cancer cells and the EPR effect in the circulatory system.

22


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1 2

3.9 In vivo anti-metastasis and anti-tumor effects of DOX-AMD-DNG

3

The metastatic property of 4T1 cells in the syngeneic tumor model in Balb/C mice is

4

well-characterized and accepted, so we used it to mimic human cancer progression and

5

metastasis in our study. To confirm the anti-cancer and anti-metastasis effect in vivo, 4T1

6

cells were xenografted into the breast fat pad of Balb/C mice and anti-cancer treatments were

7

performed when the tumors were around 50 mm3. The mice were treated with PBS, free

8

DOX, free AMD3100, DOX-DNG or DOX-AMD-DNG. As shown in Figure 10A-B,

9

encapsulation of DOX in the nanogel clearly improved effect on the tumor growth

10

suppression. The tumor weight in the DOX-AMD-DNG-treated group was much lower than

11

that of the other groups (Figure S5A). The data indicated that free DOX and DOX-DNG

12

effectively inhibited tumor growth, as revealed by the statistically significant reduction in the

13

relative tumor volume (P < 0.05). However, DOX-AMD-DNG was more effective at

14

inhibiting tumor growth (P < 0.01). DOX-AMD-DNG inhibited tumor growth more

15

efficaciously than DOX-DNG, implying that the AMD3100 modification accomplished our 23



1

goal of achieving targeted delivery to some extent. More importantly, in respect to the safety

2

of DOX-AMD-DNG for in vivo applications, no significant weight change was observed in

3

any of the treatment groups, as shown in Figure S5B. This proves that these nanocarriers are

4

biologically safe.

5 6

The anti-metastatic effect of AMD3100 was further confirmed by observing lung lesions

7

and staining lung tissue sections with H&E. In the control Balb/C mice, many metastatic

8

niches formed in the lungs, whereas mice injected with DOX-AMD-DNG had nearly no

9

niches in their lungs, as shown in Figure 10C.28 It should also be noted that the anti-

10

metastatic effect was stronger in the DOX-AMD-DNG group than in the free AMD3100

11

group, probably due to the inhibition of tumor growth by DOX, which also reduced the lung

12

metastasis. These results suggested that the co-delivery of AMD3100 and DOX can 24


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1

efficiently inhibit tumor metastasis to the lungs.

2

The anti-tumor efficacy was further evaluated by histopathological analysis of H&E-

3

stained lung and tumor sections (Figure 10D). This revealed that the tumor cells in the PBS-

4

and AMD3100-treated groups exhibited intact structures with more chromatin and

5

binucleolates, suggesting that the tumor cells were in rapid growth. In contrast, the tumor

6

cells treated with DOX formulations all exhibited significant but different degrees of tissue

7

necrosis. Furthermore, their nuclei were pyknotic. The tumors from the DOX-AMD-DNG-

8

treated group exhibited the largest necrotic areas among all the tested groups, demonstrating

9

that DOX-AMD-DNG was a more effective anti-tumor treatment than free DOX and DOX-

10

DNG. DNG with AMD3100 reduced the ratio of lung metastasis and invasive behavior of

11

breast cancer cells in the mouse xenograft model. Although DOX suppressed the tumor

12

growth, it could not completely prevent metastasis to the lung. Taken together, these data

13

indicated that DOX-AMD-DNG inhibited the growth and lung metastasis of breast cancer

14

cells synergistically.

15

25



1

4. Conclusion

2

In this study, we confirmed that CXCR4 can be used to target drug delivery using an

3

AMD3100-modified redox-responsive crosslinked Dex nanogel for treatment of metastatic

4

breast carcinoma. We evaluated the ability of the nanogel to impede metastasis and facilitate

5

CXCR4 targeting for anti-tumor drug delivery in vivo and in vitro.

6

DOX was first efficiently assembled into the crosslinked nanogel with a rather high DL

7

of 13.45%. We then modified the nanogel with AMD3100 and confirmed that the modified

8

nanogel exhibited a reduction-responsive release of DOX in vitro. The AMD3100-coated

9

DOX-DNG displayed enhanced uptake by 4T1 cells and greater cytotoxicity to 4T1 cells

10

compared with DOX-DNG due to the AMD3100-mediated targeting of CXCR4. Meanwhile,

11

CXCR4 antagonism and cell invasion assays confirmed that DOX-AMD-DNG fully inhibited

12

CXCR4 internalization and cell invasion through Matrigel. In vivo imaging and

13

biodistribution assays revealed that DOX-AMD-DNG achieved superior accumulation in

14

tumors because of the targeting property of AMD3100. Furthermore, the nanogel possessed

15

remarkable anti-cancer activity and anti-metastatic effects in 4T1 tumor-bearing Balb/C mice.

16

In summary, DOX-AMD-DNG can effectively target the tumor site and dually impede

17

cancer progression and metastasis. We predict that this bioreducible cross-linked Dex

18

nanogel, which is coated with AMD3100 and loaded with DOX, may be a safe, tumor-

19

targeting system for metastatic cancer therapy.

20 21 22 23

Acknowledgments This work was financially supported in part by the China National Science Foundation (Nos. 81373983 and 81573377) and in part by the Changjiang Scholar program.

24 26


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Biomacromolecules

1

Figure Legends

2

Scheme 1. Schematic illustration of CXCR4-targeted DOX delivery by bioreducible dextrin

3

nanogel in metastatic breast cancer.

4

Figure 1. The 1H-NMR spectra of Dex (A), Dex-COOH (B) and Dex-cys (C).

5

Figure 2. (A) Size distribution of DOX-AMD-DNG by DLS and TEM. TEM scale bar: 200

6

nm. (B) Change in particle size and morphology after incubation with 10 mM GSH for 2 h

7

detected by DLS and TEM. Scale bar: 200 nm.

8

Figure 3. In vitro release of DOX from DOX-AMD-DNG in the presence of pH 7.4 PBS with

9

10 µM GSH and pH 5.0 PBS with 10 mM GSH at 37 °C. The release behavior of free DOX in

10

pH 7.4 PBS was measured as the control. Data are presented as mean values ± SD (n = 3).

11

Figure 4. In vitro cytotoxicity of dextrin (A), free DOX, DOX-DNG and DOX-AMD-DNG

12

(B) to 4T1 cells. The different formulations were incubated with 4T1 cells at DOX

13

concentrations of 0, 0.25, 0.5, 1, 4, 8, 16 µg/ml for 24 h. (mean ± SD, n=3).

14

Figure 5. Confocal laser scanning microscopy images of 4T1 cells after treatment with (A)

15

free DOX, (B) DOX-DNG and (C) DOX-AMD-DNG for 4 h. The DOX dosage was 0.5

16

µg/ml. DOX was detected by its red fluorescence. Cell nuclei were stained with DAPI (blue).

17

The scale bar is 100 µm.

18

Figure 6. Flow cytometry analyses of 4T1 cells after different treatments. (A) Cells were

19

treated for 4 h with free DOX, DOX-DNG, DOX-AMD-DNG and DOX-AMD-DNG

20

following pretreatment with 0.6 µg/ml AMD3100. DOX concentrations were 0.25, 0.5 and 1

21

µg/ml. (B) Cells were treated for 1, 2 and 4 h with free DOX, DOX-DNG, DOX-AMD-DNG

22

and DOX-AMD-DNG after pretreatment with 0.6 µg/ml AMD3100. The DOX concentration

23

was 0.5 µg/ml.

24

Figure 7. CXCR4 antagonism. U2OS cells expressing EGFP-CXCR4 were treated as

25

follows: (A) SDF-1, (B) free AMD3100 for 30 min before incubation with 20 nM SDF-1, (C) 27



1

untreated and (D) AMD-DNG for 30 min before incubation with 20 nM SDF-1. AMD3100

2

was used as a positive control and untreated group as a negative control. Results are shown as

3

mean antagonism ± SD (n = 3).

4

Figure 8. Inhibition of cancer cell invasion of U2OS cells treated with culture medium, free

5

AMD3100, DNG and AMD-DNG for 16 h. AMD3100 was used as the positive control. The

6

bar chart shows the mean number of invading cells ± SD (n = 3).

7

Figure 9. Bioluminescence images (BLI) of 4T1 orthotopic tumors treated with Cy7-DNG

8

and Cy7-AMD-DNG at a Cy7 concentration of 200 µg/kg. Images of Cy7 fluorescence are

9

shown. White arrows indicate tumor sites.

10

Figure 10. In vivo anti-tumor and anti-metastatic effect of mice treated with saline, free

11

DOX, free AMD3100, DOX-DNG and DOX-AMD-DNG. (A) Tumor tissues excised from

12

mice in breast tumor-bearing Balb/C mice. (B) Tumor growth inhibition of mice treated with

13

the indicated formulations (n = 5). (C) Representative lungs from mice injected with different

14

formulations. Black arrows indicate the metastatic foci. (D) Histopathological (H&E) analysis

15

of tumor tissues and lungs from mice treated with the indicated formulations. Stars indicate

16

cancer cell remission. Red circles are the tumor metastases to the lung. The tumor tissues

17

were collected at day 21.

18 19

28


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1

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siRNA

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CXCR4-targeted

Nanoparticles

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Modulates

Tumor


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acquired drug resistance in liver cancer. Biomaterials 2015, 67, 194-203.

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26. Choi, K. M.; Jang, M.; Kim, J. H.; Ahn, H. J., Tumor-specific delivery of siRNA using

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protein/siRNA complexes. Biomaterials 2014, 35, (25), 7121-32.

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Lee, D. S.; Park, J. H., Gold-installed biostable nanocomplexes for tumor-targeted siRNA

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delivery in vivo. Chemical communications 2015, 51, (93), 16656-9.

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28. Zhang, C.; Gao, L.; Cai, Y.; Liu, H.; Gao, D.; Lai, J.; Jia, B.; Wang, F.; Liu, Z., Inhibition

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Scheme 1. Schematic illustration of CXCR4-targeted DOX delivery by bioreducible dextrin nanogel in metastatic breast cancer.


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Figure 1. The 1H-NMR spectra of Dex (A), Dex-COOH (B) and Dex-cys (C).



Figure 2. (A) Size distribution of DOX-AMD-DNG by DLS and TEM. TEM scale bar: 200 nm. (B) Change in particle size and morphology after incubation with 10 mM GSH for 2 h detected by DLS and TEM. Scale bar: 200 nm.


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Figure 3. In vitro release of DOX from DOX-AMD-DNG in the presence of pH 7.4 PBS with 10 μM GSH and pH 5.0 PBS with 10 mM GSH at 37 °C. The release behavior of free DOX in pH 7.4 PBS was measured as the control. Data are presented as mean values ±SD (n = 3).



Figure 4. In vitro cytotoxicity of dextrin (A), free DOX, DOX-DNG and DOX-AMD-DNG (B) to 4T1 cells. The different formulations were incubated with 4T1 cells at DOX concentrations of 0, 0.25, 0.5, 1, 4, 8, 16 μg/ml for 24 h. (mean ±SD, n=3).


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Figure 5. Confocal laser scanning microscopy images of 4T1 cells after treatment with (A) free DOX·HCl, (B) DOX-DNG and (C) DOX-AMD-DNG for 4 h. The DOX dosage was 0.5 μg/ml. DOX was detected by its red fluorescence. Cell nuclei were stained with DAPI (blue). The scale bar is 100 μm.



Figure 6. Flow cytometry analyses of 4T1 cells after different treatments. (A) Cells were treated for 4 h with free DOX, DOX-DNG, DOX-AMD-DNG and DOX-AMD-DNG following pretreatment with 0.6 μg/ml AMD3100. DOX concentrations were 0.25, 0.5 and 1 μg/ml (*P < 0.05, **P

< 0.01 and NS, vs DOX-DNG group). (B) Cells were treated for 1, 2 and 4 h with free DOX,

DOX-DNG, DOX-AMD-DNG and DOX-AMD-DNG after pretreatment with 0.6 μg/ml AMD3100 (*P < 0.05, **P < 0.01 and NS, vs DOX-DNG group). The DOX concentration was 0.5 μg/ml.


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Figure 7. CXCR4 antagonism. U2OS cells expressing EGFP-CXCR4 were treated as follows: (A) SDF-1, (B) free AMD3100 for 30 min before incubation with 20 nM SDF-1, (C) untreated and (D) AMD-DNG for 30 min before incubation with 20 nM SDF-1. AMD3100 was used as a positive control and untreated group as a negative control. Results are shown as mean antagonism ±SD (n = 3).



Figure 8. Inhibition of cancer cell invasion of U2OS cells treated with culture medium, free AMD3100, DNG and AMD-DNG for 16 h. AMD3100 was used as the positive control. The bar chart shows the mean number of invading cells ±SD (n = 3). (**P < 0.01, vs DNG treated group)


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Figure 9. Bioluminescence images (BLI) and the semi-quantative comparison of 4T1 orthotopic tumors treated with Cy7-DNG and Cy7-AMD-DNG at a Cy7 concentration of 200 μg/kg. Images of Cy7 fluorescence are shown. White arrows indicate tumor sites.



Figure 10. In vivo anti-tumor and anti-metastatic effect of mice treated with saline, free DOX, free AMD3100, DOX-DNG and DOX-AMD-DNG. (A) Tumor tissues excised from mice in breast tumor-bearing Balb/C mice. (B) Tumor growth inhibition of mice treated with the indicated formulations (n = 5). (**P < 0.01, vs saline group) (C) Representative lungs from mice injected with different formulations. Black arrows indicate the metastatic foci. (D) Histopathological (H&E) analysis of tumor tissues and lungs from mice treated with the indicated formulations. Stars indicate cancer cell remission. Red circles are the tumor metastases to the lung. The tumor tissues were collected at day 21.


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CXCR4-Targeted and Redox Responsive Dextrin Nanogel for

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