Angiopep-2-Conjugated âCoreâShellâ Hybrid Nanovehicles for

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Angiopep-2 conjugated “core-shell” hybrid nanovehicles for targeted and pH-triggered delivery of arsenic trioxide into glioma Jiaoyang Tao, Weidong Fei, Hongxia Tang, Chaoqun Li, Chaofeng Mu, Hongyue Zheng, Fanzhu Li, and Zhihong Zhu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01056 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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Molecular Pharmaceutics

1

Angiopep-2 conjugated “core-shell” hybrid nanovehicles for

2

targeted and pH-triggered delivery of arsenic trioxide into

3

glioma

4

Jiaoyang Tao1#; Weidong Fei2#; Hongxia Tang1; Chaoqun Li1; Chaofeng Mu1; Hongyue

5

Zheng3; Fanzhu Li1*; Zhihong Zhu1*

6 7

1College

8

310053, China.

9

2Department

of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou,

of Pharmacy, Women's Hospital School of Medicine Zhejiang University,

10

Hangzhou, 310006, China.

11

3Libraries

12

Hangzhou, 310053, China.

13

#These

14

*Corresponding author:

15

Fanzhu Li, College of Pharmaceutical Science, Zhejiang Chinese Medical University,

16

Gaoke Road, Fuyang District, Hangzhou, 311402, China.

17

Tel.: +86-571-61768130, Fax: +86-571-86613607, E-mail: [email protected].

18

Zhihong Zhu, College of Pharmaceutical Science, Zhejiang Chinese Medical

19

University, Gaoke Road, Fuyang District, Hangzhou, 311402, China.

20

Tel.: +86-18809896807, E-mail: [email protected].

of Zhejiang Chinese Medical University, Zhejiang Chinese Medical University,

authors contributed equally to the work.

21

ACS Paragon Plus Environment

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ABSTRACT

2

The poor capability of drugs to permeate through blood brain barrier (BBB) and

3

further release inside glioma greatly limit the curative effects of glioma

4

chemotherapies. In this study, we prepared angiopep-2 conjugated liposome-silica

5

hybrid nanovehicles for targeted delivery and increased the permeation of arsenic

6

trioxide (ATO) in glioma. Polyacrylic acid (PAA) was grafted on mesoporous silica

7

nanoparticles (MSN) for pH-sensitive release and supporting lipid membrane. The

8

prepared “core-shell” nanovehicles (ANG-LP-PAA-MSN) were characterized with

9

uniform size, high drug loading efficiency (8.19 ± 0.51%), and superior pH-sensitive

10

release feature. From the experiments, the enhanced targeted delivery of ATO by

11

ANG-LP-PAA-MSN

12

improvement of transport, enhanced cellular uptake and apoptosis in vitro. In addition,

13

the pharmacokinetic study was creatively carried out through the blood-glioma

14

synchronous microdialysis and revealed that the half-life (t1/2) of blood and glioma

15

tissue in the ANG-LP-PAA-MSN@ATO treatment group was extended by 1.65 and

16

2.34 times compared with the ATO solution group (ATO-Sol). The targeting

17

efficiency of ANG-LP-PAA-MSN@ATO (24.96%) was dramatically stronger than

18

that of the ATO-Sol (5.94%). Importantly, ANG-LP-PAA-MSN@ATO had higher

19

accumulation (4.6 ± 2.6% ID per g) in tumor tissues and showed a better therapeutic

20

efficacy in intracranial C6 glioma bearing rats. Taken together, the blood-glioma

21

synchronous microdialysis was successful used to the pharmacokinetic study and

22

real-time monitored drug concentrations in blood and glioma; ANG-LP-PAA-MSN

(ANG-LP-PAA-MSN@ATO)


was

evidenced

by

the

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1

could be a promising targeted drug delivery system for glioma therapy.

2 3

KEYWORDS: microdialysis; arsenic trioxide; pH-triggered MSN; phospholipid

4

coated; angiopep-2; glioma

5 6

INTRODUCTION

7

Glioma, the most common malignant primary brain tumor, often showed the

8

characterization of invasive growth, poor prognosis and high recurrence. In spite of

9

positive therapies like surgery, radiotherapy and chemotherapy are taken before the

10

deterioration, the median life expectancy of glioma patients is only 14−20 months,

11

emphasizing the urgent needs for novel therapeutic manners.1 Therapy choices for

12

glioblastoma still restrained to some extent because of the drug resistance of glioma

13

cells to chemotherapy as well as the aporia of delivery therapeutics into the brain by

14

passing the blood brain barrier (BBB).2, 3 At the same time, serious adverse reactions

15

such as liver damage, gastrointestinal discomfort and leucopenia caused by systemic

16

administration of chemotherapy drugs cannot be ignored.4,

17

antitumor drugs and designing more efficient delivery strategies become an urgent

18

need for clinical treatment of glioma.

5

Thus, finding new

19

Arsenic trioxide (ATO), as an active ingredient of traditional Chinese medicine,

20

is a frontline drug for the therapy of acute promyelocytic leukemia (APL) for

21

decades.6 Recently, extensive researches have demonstrated that ATO could be used

22

as a new and valid antineoplastic for treating glioma by inhibiting cell proliferation



1

and inducing apoptotic effects.7, 8 However, its adverse reactions, high renal clearance

2

rate9, 10 and poor permeability crossing the BBB hinder its application in the treatment

3

of glioma. To realize this purpose, a variety of drug delivery nanocarriers have been

4

used to deliver inorganic ATO, such as liposomes,11 polymer nanoparticles12 and

5

mesoporous silica nanoparticles (MSN).13 However, the organic nanocarriers were

6

incompatible with chemical compositions which caused by the low drug loading

7

capability and instability during storage. Meanwhile, MSN could load the drug

8

efficiently and release them sustainedly due to its highly ordered mesoporous

9

structure, extremely high specific surface area and pore volume.14 Nevertheless, MSN

10

based ATO nano-formulations existed serious burst drug release during systemic

11

circulation.

12

Liposome-silica hybrid nanocarriers with the exception of the properties of

13

inorganic MSN, of which functional liposomes, are suitable for ATO loading as the

14

following reasons: 1) the phospholipid bilayer reduces the burst release of drugs by

15

blocking mesopores and increases the affinity between the drug delivery system and

16

the cell membrane; 2) the targeted ligands inserted in the phospholipid layer can

17

significantly increase drugs accumulation in tumor site. It is reported that after

18

delivered with liposome-silica hybrid nanovehicles, ATO could treat hepatic

19

carcinoma more efficiently.15

20

To further achieve the specific drug release at the tumor site, the polyacrylic acid

21

(PAA), a most common used pH-responsive material with low toxicity and good

22

biocompatibility16,

17

is considered to graft on the outer surface of MSN. The


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1

hydrophobic interactions and Van Der Waals between silica core and liposomal shell,

2

which supplied by the organic carbon chain of PAA, would enhance the constancy of

3

the “core-shell” structure in blood circulation and sustain the drug release in a

4

controllable and efficacious manner.15 Meanwhile, lipoprotein receptor-related protein

5

(LRP) receptor expressed on both glioma cells and brain endothelial cells.18,

6

Angiopep-2 (TFFYGGSRGKRNNFKTEEY, 2.4 kDa MW), a specific ligand of LRP

7

receptor, would be modified on the lipid bilayer to increase the accumulation of ATO

8

in the glioma.

19

9

In this paper, we would introduce a kind of hybrid nanovehicles consisting of

10

PAA modified mesoporous silica core and an angiopep-2-modified liposomal shell to

11

achieve targeted delivery and on-demand release of ATO for brain glioma therapy

12

(Figure 1). The morphology and structure of nanoparticles were characterized. The

13

properties of drug release, cellular uptake and BBB permeability were demonstrated

14

by the in vitro evaluations and the therapeutic efficacy of targeted delivery of ATO by

15

nanocarriers (ANG-LP-PAA-MSN@ATO) on orthotopic glioma bearing rats and

16

distribution into glioma were investigated. What’s more, the targeting efficiency and

17

pharmacokinetics of the nanodrug in blood and tumor site were evaluated with the

18

blood-glioma synchronous microdialysis technique.



Page 6 of 38

1 2

Figure 1. Schematic illustration for the synthesis and preparation of ANG-LP-PAA-MSN@ATO for

3

glioma therapy.

4 5

EXPERIMENTAL SECTION

6

Materials. Arsenic trioxide (ATO, 90%) was obtained from Alfa Aesar (Shanghai,

7

China). Tetraethyl orthosilicate (TEOS, 98%), cetyltrimethylammonium bromide

8

(CTAB, 99%), N,N-Dimethylformamide (DMF) were obtained from Aladdin Co.

9

(Shanghai, China). Polyacrylic acid (PAA) was purchased from Sigma-Aldrich (St.

10

Louis, USA). Angiopep-2 (TFFYGGSRGKRNNFKTEEYC) was synthesized and

11

validated

12

1,2-Distearoyl-sn-glycero-3-phosphatidylcholine

13

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol-

14

2000] (DSPE-PEG2000-MAL) were purchased from A.V.T Co. (Shanghai, China).

15

4',6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime Biotechnology

by

the

Chinese

Peptide

Co. (DSPC),


(Hangzhou, cholesterol

China). and

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1

(Shanghai, China). D-Hank’s buffer solution was purchased from Gibco BRL

2

(Gaithersberg MD, USA). All the other chemicals were analytical grade, and ultrapure

3

water was utilized throughout this study via a Millipore water purification system.

4

Cell culture and animals. Human brain microvascular endothelial cells (HBMEC)

5

and C6 glioma cells were obtained from the Laboratory Animal Research Center,

6

Zhejiang Chinese Medical University (Hangzhou, China) and cultured in DMEM with

7

10% FBS and 1% penicillin-streptomycin with a fully humidified atmosphere at

8

37 °C with 5% CO2.

9

Sprague-Dawley (SD) rats (weight 200 ± 10 g) were provided by the Laboratory

10

Animal Research Center, Zhejiang Chinese Medical University (Hangzhou, China).

11

All experiments were conducted in accordance with the guidelines of the care and use

12

of animals as framed and supervised by Zhejiang Chinese Medical University.

13

Preparation of NH2-MSN. The amino functionalized MSN (NH2-MSN) were

14

synthesized with improved Stober method based on a previously reported method.20

15

In brief, 0.3 g CTAB was added in ultrapure water. The pH value of the solution was

16

about 11.5 by added to NaOH (2 mol/L). APTES (0.5 mL) and TEOS (1 mL) were

17

added dropwise to the solution for 2 h after vigorous stirring and heated up to 80 °C

18

for 0.5 h. The obtained particles were centrifuged (20,000 rpm, 30 min). To remove

19

the surfactant CTAB, the product was washed twice with acidic ethanol and ultrapure

20

water, finally centrifuged and obtained by freeze-dried for further use.

21

Drug loading. For the loading of the anti-cancer drug ATO, 40 mg MSN was added

22

in 10 mL of ATO solution (1 mg/mL), stirring for 2 h. Thereafter, ATO-loaded



Page 8 of 38

1

NH2-MSN (MSN@ATO) were centrifugated (20,000 rpm, 30 min) and washed three

2

times with ultrapure water, then collected and freeze-dried.

3

Synthesis of PAA-MSN. To obtain the PAA capped MSN (PAA-MSN), 20 mg PAA

4

was added in 20 mL DMF containing 20 mg NH2-MSN under stirring at 100 °C for 2

5

h. The products were centrifuged (20,000 rpm, 30 min), washed three times with

6

ethanol and ultrapure water alternately and dried under vacuum.

7

Preparation of LP-PAA-MSN and ANG-LP-PAA-NSN. To synthesis of

8

angiopep-2 conjugated phospholipid (DSPE-PEG2000-angiopep-2), DSPE-PEG2000-

9

MAL was reacted with angiopep-2 (the molar ratio of maleimide to angiopep-2 was

10

3:1) in PBS (pH 7.4) for 24 h at 25 °C. The resultant product was purified using the

11

dialysis method for 48 h to remove the free angiopep-2 and DSPE-PEG2000-Mal.

12

Finally, the products were freeze-dried for further use.

13

Liposome-silica hybrid nanovehicles (LP-PAA-MSN) were prepared according

14

to modified thin film hydration method.21 The liposome carriers composed of

15

DSPC/Chol/DPSE-PEG2000 (65:25:10, w/w/w) were dissolved in chloroform. 10 mg

16

PAA-MSN was distributed by stirring and evaporated under negative pressure at

17

45 °C. The dried film was hydrated in ultrapure water at 50 °C for 10 min and

18

collected.

19

(ANG-LP-PAA-MSN)

20

DSPC/Chol/DPSE-PEG2000/DPSE-PEG2000-angiopep-2 (65:25:8:2, w/w/w/w). The

21

drug-loaded

22

(PAA-MSN@ATO), and fabricated by the same method.

Angiopep-2

liposomes

conjugated were

were

liposome-silica prepared

prepared

by


hybrid at

ATO-loaded

nanovehicles ratio

of

PAA-MSN

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1

Characterization of nanoparticles. Nanoparticles were visualized via transmission

2

electron microscopy (TEM) images on a transmission electron microscopy (S4800,

3

Hitachi, Japan). ζ-potential, polydispersity index (PDI) and size diameter of the

4

samples were obtained by Dynamic Light Scattering (DLS) (Malvern ZEN 3690, UK).

5

Nitrogen sorption isotherms were obtained using a micromeritics ASAP-2020

6

sorptometer (Micromeritics, USA) after degassing of the samples at 150 °C,

7

calculated the pore size distribution by the Brunauer Emmett Teller (BET) method

8

and the specific surface area by the Barrett Joyner Halenda (BJH) method.

9

Thermogravimetric analysis (TGA) was carried out on a Thermal Gravimetric

10

Analyzer (NETZSCH STA 449 F3, Germany) by heating to 800 °C. Powder X-ray

11

pattern (XRD) was obtained to study mesoporous structure by a Bruker D4 X-ray

12

diffractometer (Bruker D8 advance, Germany). The infrared signature of NH2-MSN

13

and PAA-MSN was recorded with a Fourier transform infrared (FTIR)

14

spectrophotometer (Nicolet IS500, USA).

15

Stability study. Firstly, we prepared FITC labeled nanoparticles. Briefly, the mixture

16

of FITC (2 mg) and NH2-MSN (50 mg) was added in DMF (10 mL) in dark at room

17

temperature for 12 h and stirred. Then, the FITC labeled NH2-MSN (MSN@FITC)

18

were centrifuged, then washed with ethanol three times. The PAA-modified and

19

lipid-coated

20

PAA-MSN@FITC, LP-PAA-MSN@FITC and ANG-LP-PAA-MSN@FITC. 3

21

mg/mL

22

LP-PAA-MSN@FITC and ANG-LP-PAA-MSN@FITC) were dispersed in normal

process

what

FITC-labeled

we

mentioned

nanocarriers

above

were

(MSN@FITC,


used

to

prepare

PAA-MSN@FITC,


Page 10 of 38

1

saline. The size diameter was recorded at 0.5, 1, 1.5, 2, 3, 4, 8, 12, 18, 24 and 48 h.

2

The FITC-labeled nanocarriers were observed the aggregation state using UV light

3

(365 nm) at the start and the end of the study.

4

In vitro ATO release study. To evaluate the in vitro ATO release, MSN@ATO,

5

PAA-MSN@ATO, ANG-LP-PAA-MSN@ATO were investigated using the dialysis

6

bag method (7 kDa MW). ATO formulations were immersed in 100 mL of PBS (pH

7

7.4, 6.0 and 5.0) at 37 °C. Samples were withdrawn at designated times, and replaced

8

with the same fresh dissolution medium and analyzed via inductively coupled plasma

9

emission spectrum (ICP, 6300, Thermo Electron Corporation, USA).

10

Cellular uptake and intracellular disposition. HBMEC cells and C6 cells were

11

cultured in 6-well plates at s seeding density of 5 × 105 cells per well and cultured for

12

12 h at 37 °C, then treated with culture medium or the FITC loaded

13

nanoparticles-containing

14

LP-PAA-MSN@FITC

15

concentration of 3 mmol/L) for 4 h, washed with PBS three times, and harvested.

16

Eventually, the fluorescence intensity was measured by flow cytometer (Millipore

17

Guava, Germany).

culture and

medium

(MSN@FITC,

ANG-LP-PAA-MSN@FITC)

PAA-MSN@FITC, (equivalent

FITC

18

Confocal laser scanning microscopy (LSCM, Zeiss LSM880, Carl Zeiss,

19

Germany) was detected intracellular localization of the FITC-labeled nanoparticles in

20

HBMEC cells and C6 cells. HBMEC cells and C6 cells were incubated in the sterile

21

glass bottom dishes at density of 1×105 cells per well and cultured for 12 h.

22

Subsequently, cells were treated with the FITC loaded nanoparticles-containing


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1

culture medium (equivalent FITC concentration of 3 mmol/L) for 4 h. Cells were

2

washed with PBS three times, then fixed with 4% paraformaldehyde and stained with

3

DAPI.

4

Cytotoxicity evaluation. The cytotoxicity of blank nanoparticles (NH2-MSN,

5

PAA-MSN, LP-PAA-MSN and ANG-LP-PAA-MSN) and ATO formulations on both

6

HBMEC cells and C6 cells was assessed by employing the MTT assay. In a nutshell,

7

the cells were cultured in a 96-well plate at a seeding density of 5 × 103 cells per well

8

in 200 μL culture medium and cultured for 12 h. Then, cells were treated with

9

drug-free carriers or ATO formulations with a series of concentrations for 48 h.

10

Subsequently, the culture medium was aspirated off and cells were incubated with

11

PBS containing 100 μL 0.5 mg/mL of MTT for 4 h. After the MTT was discarded,

12

formazan crystals were dissolved in 150 mL DMSO, and the absorbance of each

13

group was measured at 570 nm with a microplate reader (Synergy TM2, BIO-TEK

14

Instruments Inc. USA).

15

BBB penetration in vitro. BBB model in vitro was constructed according to previous

16

described,22 the HBMEC cells (1 × 105 cells per well) were seeded into the transwell

17

inserts (polycarbonate 12-well transwell membrane of 3 μm mean pore size, Corning,

18

NY, USA) and the culture medium was replaced every other day. The completeness

19

of BBB model was assessed with transendothelial electrical resistance instrument

20

(Millicell-ERS-2, Millipore, USA) in accordance with the TEER value (with the

21

threshold value of 250 Ω/cm2).

22

To investigate the capability of ATO formulations across the BBB, ATO-Sol,



PAA-MSN@ATO,

LP-PAA-MSN@ATO

Page 12 of 38

1

MSN@ATO,

2

ANG-LP-PAA-MSN@ATO were added into the corresponding insert, respectively.

3

The culture medium was replaced with D-Hank’s buffer solution as a transport

4

medium. 200 μL of buffer solution was taken out from the basolateral compartments

5

at 1, 2, 4, 6 and 8 h after treatment, then replaced with an equal volume of fresh

6

medium immediately. The BBB transport ratios of ATO were assessed by inductively

7

coupled plasma mass spectrometry (ICP-MS, 7500ce, Agilent, USA).

8

Cell cycle analysis. C6 cells were treated with free ATO or ATO-formulations

9

containing culture medium (MSN@ATO, PAA-MSN@ATO, LP-PAA-MSN@ATO

10

and ANG-LP-PAA-MSN@ATO) as control for 48 h. After collecting the cells, we

11

fixed them with 70% ethanol at 4 °C for 8 h. Then, the ethanol was removed. After

12

incubated with DNase-free RNase A (1 mg/mL) for 0.5 h at 37 °C, the cells were

13

stained with 0.1% of Triton X-100 (0.1 mL, which contains 0.02 mg/mL of PI), then

14

analyzed via flow cytometer (Millipore Guava, Germany). The BBB model was

15

established for analyzing the C6 cell cycle after the drug-containing formulation

16

transport. Briefly, the in vitro BBB model established, C6 cells were collected in the

17

basolateral compartment after adding the formulations into the upper chamber, treated

18

for 48 h, followed by the mentioned steps above for analysis.

19

Pharmacokinetic Study. A rat model bearing orthotopic glioma was established

20

according to the reported method.23 Rats were anaesthetized with 3% pentobarbital

21

(i.p. 1.5 mL/kg) and secured in a stereotaxic frame (Bioanalytical Systems, West

22

Lafayette, IN, USA). Then, the cranium was exposed a midline sagittal incision.


and

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1

Afterward, a burr hole (diameter: 1.5 mm) was drilled at a special location (3 mm

2

lateral to the bregma and 1 mm anterior to the right coronal suture). Around 1×106 C6

3

glioma cells in 10 μL PBS were stereotaxically injected into the right forebrain at a

4

depth of 5 mm. Using the bone wax covering, the scalp incision was closed.

5

After recovering, according to our previous study,24 a hole (0.5 mm) was drilled

6

at the skull of rats (0.2 mm anterior to bregma and 3.2 mm lateral to the midsagittal

7

suture). Chronic brain microdialysis guide cannulas (MD-2251, BAS, West Lafayette,

8

IN, USA) with stylet in place was implanted in the left striatum with a depth of 7.0

9

mm ventrally from the dura. The guide cannula was fastened to the cranium with skull

10

screws and dental acrylic cement.

11

Rats recovered after surgery for six days, the blood microdialysis probe (15 kDa

12

MW) was inserted into the guide cannula of the jugular vein, and Ringer’s solution

13

and artificial cerebral spinal fluid were used to perfuse the blood and brain probe at a

14

flow rate of 2.0 μL/min, respectively. Then, ATO formulations (ATO-Sol,

15

MSN@ATO,

16

ANG-LP-PAA-MSN@ATO) were injected via tail vein (1 mg/kg) after equilibrating

17

the probe for 1 h in awake rats. The dialyzate samples were collected every 0.5 h in

18

the first 6 h and every 1 h from 6 h to 12 or 24 h. And the samples were stored at

19

-20 °C and analyzed by ICP-MS. ICP-MS working conditions were as follows: the

20

cooling gas flow 15 L/min, RF power 1500 W, carrier gas flow 0.81 L/min, plasma

21

gas flow 15 L/min, auxiliary gas flow 0.22 L/min, diameter sampler 1.0 mm,

22

sampling depth 8.1 mm, diameter of skimmer 0.4 mm, sweeping times and main runs

PAA-MSN@ATO,

LP-PAA-MSN@ATO


and


Page 14 of 38

1

three times respectively, nebulizer was 100 μL quartz with core flow. The calibration

2

curve was constructed over a range of 1-50 μg/L in plasma (Y=1875.3 C + 1205.7,

3

r=0.9912) and in glioma (Y=1639.6 C + 797.5, r=0.9938). The recovery, inter-day

4

and intra-day precision values were within acceptable range. The working conditions

5

were similar to methods reported previously.20

6

The relative drug targeting efficiency (Te) was calculated with the formula:

7

Te = AUCglioma/AUCblood

8

In vivo biodistribution. In vivo biodistribution analysis was conducted on the

9

intracranial C6 glioma-bearing rats after seven days recovering, injected with saline or

10

ATO

11

LP-PAA-MSN@ATO and ANG-LP-PAA-MSN@ATO) at a dose of 1 mg/kg via tail

12

vein. The isolated tissues of the rats in each group (n = 5) were excised 24 h after

13

injection and lysed in 2 mL mixed hydrogen peroxide and nitric acid, assessed by

14

ICP-MS.

15

In vivo anti-tumor study. The intracranial C6 glioma-bearing rats were randomly

16

divided into six groups (11 rats/group) after seven days recovering, injected with

17

saline or ATO formulations (ATO-Sol, MSN@ATO, PAA-MSN@ATO and

18

ANG-LP-MSN@ATO) at a dose of 1 mg/kg via tail vein every two days and body

19

weight was recorded. At the 17th day, the brain tissues of five rats in each group were

20

collected, dissected and the length and width of the glioma were measured to calculate

21

the volume of glioma according to equation:

22

formulations

(ATO-Sol,

MSN@ATO,

V = (length × width2)/2


PAA-MSN@ATO,

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1

Then the brain tissues were fixed in formalin, paraffin embedded and stained

2

with hematoxylin and eosin (H&E) to assess the apoptosis of tumor cells. The

3

remanent six rats of each group were recorded the survival time.

4

Statistical analysis. All data of experiments was expressed as the mean ± standard

5

deviation (SD). Statistical significance was performed by one-way ANOVA with

6

SPSS software (version 23.0, SPSS, Inc., Chicago, IL, USA). The survival data was

7

carried out through Kaplan-Meier curves and was analyzed using the logrank test. In

8

all studies, P < 0.05 was considered to be statistically significant.

9 10

RESULTS AND DISCUSSION

11

Synthesis and characterization of NH2-MSN, PAA-MSN, LP-PAA-MSN and

12

ANG-LP-PAA-MSN. The amino modified MSN (NH2-MSN) with uniform size were

13

prepared by co-condensation method as we had reported (Figure 2A).20 The size and

14

PDI of NH2-MSN detected via dynamic light scattering (DLS) were 128.2 ± 2.8 nm

15

and 0.021 ± 0.012 (Table 1). The amino groups of NH2-MSN provided positive

16

charge to absorb anionic drugs and offered functional groups for further reaction with

17

the carboxyl group of PAA which will dissociate in tumor microenvironment.25

18

Therefore, PAA was grafted on the surface of NH2-MSN (PAA-MSN). The size of

19

PAA-MSN became bigger (about 133 nm). The outer surface of PAA-MSN became

20

rough and the ordered mesoporous structure was disappeared compared with

21

NH2-MSN (Figure 2B). The FT-IR spectra of NH2-MSN and PAA-MSN were shown

22

in Figure 3D. The presented adsorption peaks appeared at 1563.17 cm-1 and 1728.57



Page 16 of 38

1

cm-1 due to the stretching vibration of C=O of acylamino carboxylic, which indicated

2

that PAA had been conjugated on the surface of nanocarriers successfully.26,

3

better

4

adsorption-desorption measurements and XRD analysis were adopted (Figure 3A-B).

5

The BET isotherm of the NH2-MSN showed the type IV of N2 adsorption-desorption

6

patterns and the peak of pore size (about 2.45 nm) of NH2-MSN indicated

7

mesoporous were regular and uniform.28 The pore size of NH2-MSN was 2.38 ± 0.26

8

nm. The cumulative pore volume and the specific surface area of NH2-MSN were 1.14

9

± 0.11 cm3/g and 1084.68 ± 30.67 m2/g. After modified with PAA, the cumulative

10

pore volume and the specific surface area of the carriers reduced to 1.11 ± 0.09 cm3/g

11

and 654.87 ± 26.53 m2/g. Meanwhile, the pore volume, the peak of pore size and the

12

diffraction peak of PAA-MSN were nearly disappeared in PAA-MSN on account of

13

the pore-plugging effect of PAA. These above changes of nanocarriers may be caused

14

by the plugging of PAA, which enable the controlled drug release successfully.20

investigate

the

structure

of

NH2-MSN

and

PAA-MSN,

27

the

To N2

15 16 17

Table 1 The size, polydispersity index (PDI) and zeta potential of NH2-MSN, PAA-MSN,

18

LP-PAA-MSN, ANG-LP-PAA-MSN. Sample

Size (nm)

PDI

Zeta potential (mV)

NH2-MSN

128.2±2.8

0.021±0.012

40.17±1.86

PAA-MSN

133.1±1.3

0.099±0.027

-27.87±0.45

LP-PAA-MSN

139.9±2.2

0.145±0.026

-18. 60±0.72

ANG-LP-PAA-MSN

141.6±3.6

0.153±0.027

-13.93±0.46


Page 17 of 38 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 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure

3

ANG-LP-PAA-MSN (D).

2.

TEM

images

of

NH2-MSN

(A),

PAA-MSN

(B),

LP-PAA-MSN

(C),

and

4

Liposome-silica hybrid nanovehicles with a “core-shell” structure are considered

5

as ideal drug carriers for reinforcing antitumor effects of ATO. In our previous study,

6

chlorodimethyloctadecylsilane was bounded on the surfaces of hollow silica

7

nanoparticles for supporting the phospholipid bilayer, which achieved long-term

8

circulation in body and enhanced the affinity between cell membrane and drug

9

delivery

systems.15

In

this

study,

PAA

was

used

instead

of

10

chlorodimethyloctadecylsilane to act as skeleton for bracing phospholipid bilayer. To

11

attain targeted delivery, the angiopep-2 peptides were bound to the phospholipid

12

bilayer. In the

13

characteristic peaks between 6.60 and 7.20 ppm of angiopep-2 suggested that peptides

14

had been bound to the terminal of DPSE-PEG2000 (Figure 3G). And the angiopep-2

1H-NMR

spectrum of DSPE-PEG2000-ANG, the corresponding



1

conjugating density on the nanovehicles was about 1.9%. The nonspecific

2

phospholipid bilayer was composed of DSPC, DSPE-PEG2000 and cholesterol, while

3

the targeting composition was constructed by phospholipid bilayer containing

4

DSPE-PEG2000-ANG.

5

In the TEM images (Figure 2C-D), LP-PAA-MSN and ANG-LP-PAA-MSN

6

were spherical with relatively uniform size and good dispersibility. Intact liposomal

7

shell with thickness of ~7 nm appeared on LP-PAA-MSN and ANG-LP-PAA-MSN.

8

The mean particle size of LP-PAA-MSN and ANG-LP-PAA-MSN increased to about

9

140 nm, which was suitable for endocytosis by brain capillary endothelial cells.29, 30

10

The further decreased pore size, specific surface area and the XRD peak of

11

LP-PAA-MSN and ANG-LP-PAA-MSN were attributed to the capping of

12

nanocarriers by the lipid membrane (Figure 3A-B).

13

During the storage, the nanoparticles were prone to aggregation and adsorption

14

because of high surface energy.31 The stability experiments were investigated by

15

measuring the changes of size in normal saline and observed using UV light (Figure

16

3E-F). The size distribution of the lipid-coated nanocarriers kept uniform within 48 h,

17

while MSN@FITC and PAA-MSN@FITC were gathered with an intense green

18

fluorescence at the bottom of vials. This was due to the encapsulation of lipid vesicle

19

reduced the specific surface energy of the mesoporous material and prevented them

20

from aggregation in saline solution.


Page 18 of 38

Page 19 of 38 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 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 3 N2 adsorption-desorption isotherm and pore size distribution (A), XRD patterns (B), TGA

3

curves(C), FT-IR spectra (D), stability curve (E) and dispersion state (F) of NH2-MSN, PAA-MSN,

4

LP-PAA-MSN and ANG-LP-PAA-MSN. 1H-NMR spectra (G) of DSPE-PEG2000-MAL, angiopep-2

5

and DSPE-PEG2000-angiopep-2.

6

Drug loading and in vitro release study. Drug loading mechanism was primarily

7

based on the electrostatic interaction between NH2-MSN and arsenite ion. NH2-MSN

8

could contain the loading efficiency of 10.07% ATO estimated by TGA because the

9

nanocarriers were primarily made up of SiO2 with high thermal steady (Figure 3C).32

10

The DL% of MSN@ATO as monitored by ICP was 11.06 ± 0.43%, which was a little

11

larger than that detected by TGA. By measuring the ATO in the supernatant during

12

washing, the ATO loss during the procedure was negligible. After conversion, the DL%

13

of LP-PAA-MSN@ATO and ANG-LP-PAA-MSN@ATO was 8.62 ± 0.26% and

14

8.19 ± 0.51%. Compared with organic carriers, the ATO loading ability of

15

ANG-LP-PAA-MSN was found to be higher than that in liposomes (~1.85%) and



1

polymersomes (~5.3%).33, 34

2

To assess the pH triggered and the sustained release ability of “core-shell” hybrid

3

nanovehicles, in vitro drug release experiments were performed in a physiological

4

environment. The accumulative drug release kinetics curves of MSN@ATO,

5

PAA-MSN@ATO and ANG-LP-PAA-MSN@ ATO in PBS at different pH medium

6

were showed in Figure 4. All MSN-based nano-formulations displayed sustained

7

release action for the drug diffusion from the mesoporous structure gradually,

8

compared with the release amount of ATO-Sol (91.31 ± 1.72%) in 2 h. ATO released

9

from MSN@ATO was about 82.96 ± 2.51% in 48 h. It was worth noting that about

10

50.71 ± 1.11% drug release in MSN@ATO in the earlier 2 h. The quick drug release

11

was resulted from wake bonding between ATO and the surface of the nanocarriers

12

through electrostatic attraction and Van der Waals Force.35 After grafted with PAA,

13

the burst release ratio of nanodrug further reduced to 25.83 ± 1.73% in pH 7.4. The

14

reason was that the PAA had acted as a barrier against the escape of encapsulated

15

cargos. The drug release of ATO from MSN@ATO was 85.70 ± 0.50%, 83.04 ± 0.93%

16

and 80.14 ± 0.96% at pH 5.0, 6.0, 7.4 in 48 h, respectively. Mildly pH-triggered

17

release effect was investigated in MSN@ATO, because of amino groups which could

18

provide and bind protons to carry out the variation of pH dependence.36 Additionally,

19

ATO in acid water had an increasing solubility which also promoted the faster release

20

of ATO from MSN@ATO in low pH. The accumulative release of ATO from

21

PAA-MSN@ATO was up to 77.32 ± 2.06% and 61.99 ± 1.61% at pH 5.0, 6.0 in 48 h,

22

respectively, while only 49.54 ± 0.62% of ATO was monitored at pH 7.4. The drug


Page 20 of 38

Page 21 of 38 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 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

release from PAA-MSN@ATO revealed significant pH dependence and enhanced

2

with the decline of pH value. Because the mesoporous structure was capped by PAA,

3

PAA was protonized ultimately and spread into the PBS solution, which would

4

exposure the pore of MSN with the decline of pH value. As a result, more ATO could

5

be released under low pH value.37 ANG-LP-PAA-MSN@ATO possessed identical

6

releasing profiles as PAA-MSN@ATO. This feature was key for ATO delivery to

7

glioma, owing to minimizing the premature released drug during the distribution, and

8

the

9

nanomedicines was an excellent feature in the application for cancer therapy.

characteristic

of

pH-responsive

and

enhanced

sustained-release

from

10 11

Figure 4 In vitro release profiles of ATO in PBS (pH 5.0, 6.0, 7.4) from ATO-Sol and MSN@ATO

12

(A), PAA-MSN@ATO (B), ANG-LP-PAA-MSN@ATO (C) (n=3).

13

Cellular uptake and intracellular disposition. The cellular uptake of FITC-labeled

14

nanovehicles

15

ANG-LP-PAA-MSN@FITC) into HBMEC cells and C6 cells was investigated by

16

flow cytometry. As shown in Figure 5A-B, compared with PAA-MSN@FITC, the

17

fluorescence intensity of LP-PAA-MSN@FITC and ANG-LP-PAA-MSN@FITC was

18

higher significantly after incubated for 4 h. The fluorescence intensity of

19

ANG-LP-PAA-MSN@FITC

(PAA-MSN@FITC,

was

2.16-fold

LP-PAA-MSN@FITC

stronger


than

and

that

of


Page 22 of 38

1

LP-PAA-MSN@FITC, and the fluorescence intensity of LP-PAA-MSN@FITC was

2

1.67-fold stronger than that of PAA-MSN@FITC in C6 cells. Meanwhile, the

3

fluorescence intensity of ANG-LP-PAA-MSN@FITC was 2.56-fold higher than that

4

of LP-PAA-MSN@FITC in HBMEC cells. These results demonstrated that the lipid

5

bilayer enhanced the appetency between drug delivery systems and cell membranes,

6

thus improving cellular uptake by contact-facilitated drug delivery.38 This

7

nanovehicles possibly fused with cell membranes through the pinocytosis of cells.39, 40

8

However, the outer surface of PAA-MSN@FITC was modified by the organic carbon

9

chain

which

had

poor

affinity

with

cell

membranes

compared

to

10

LP-PAA-MSN@FITC. The angiopep-2 ligand could target HBMEC cells and C6

11

cells via specific recognition, and accomplish targeted drug delivery. To determine

12

the cellular localization of nanocarriers, after C6 cells incubated for 4 h, the

13

fluorescence signal of ANG-LP-PAA-MSN@FITC was dramatically stronger

14

compared

15

LP-PAA-MSN@FITC and ANG-LP-PAA-MSN@FITC was appeared the similar

16

intensity in HBMEC cells (Figure 5C). These findings were consistent with the flow

17

cytometric analysis data.

with

that

of

LP-PAA-MSN@FITC,


while

the

signal

of

Page 23 of 38 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 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 5 Flow cytometry profiles of HBMEC cells (A) and C6 cells (B) were incubated with

3

PAA-MSN@FITC, LP-PAA-MSN@FITC or ANG-LP-PAA-MSN@FITC containing medium (3 mM

4

FITC) for 4 h. Confocal images (C) of HBMEC cells and C6 cells incubated for 4 h with

5

PAA-MSN@FITC, LP-PAA-MSN@FITC or ANG-LP-PAA-MSN@FITC containing medium. Cell

6

nuclei were stained with DAPI (blue).

7

In vitro cytotoxicity. To assess the safety of functionalized MSN, nanocarriers were

8

incubated with HBMEC and C6 cells for 48 h. As shown in Figure 6A-B, all

9

nanoparticles were demonstrated good biocompatibility and low toxicity with high

10

cell viabilities more than 85% at concentrations below 10 μg/mL. NH2-MSN revealed

11

higher cytotoxicity with the increment of the concentration. About 30.59 ± 2.39% of

12

HBMEC cells and 33.93 ± 1.69% of C6 cells were inhibited by NH2-MSN at a high



Page 24 of 38

1

concentration of 100 μg/mL during 48 h. The mechanism was perhaps attributed to

2

concentration-dependent hemolytic effect and the positive charge. The cell viability

3

incubated with PAA-MSN was higher than that of cells incubated with NH2-MSN. It

4

showed that the organic carbon chain bonded the surface of NH2-MSN could reduce

5

the cytotoxicity by masking the naked amino groups.41 The lipid-encapsulated

6

formulations (LP-PAA-MSN and ANG-LP-PAA-MSN) were enhanced the cell

7

viability of because of increased biocompatibility by imitating the component of cell

8

membranes and simulating the “protocell-like” structure.

9

To assess the cytotoxicity of drug-loaded formulations, HBMEC cells and C6

10

cells were cultured with free ATO, NH2-MSN@ATO, PAA-MSN@ATO,

11

LP-PAA-MSN@ATO and ANG-LP-PAA-MSN@ATO for 48 h, respectively. As

12

shown in Figure 6C-E, the inhibition of cells was reduced with the decrease of ATO

13

concentrations. The half maximal inhibitory concentration (IC50) values of ATO-Sol,

14

MSN@ATO,

15

ANG-LP-PAA-MSN@ATO were 6.41 ± 0.11 mM, 9.26 ± 0.29 mM, 8.28 ± 0.27 mM,

16

7.07 ± 0.13 mM and 5.56 ± 0.24 mM, respectively. The IC50 of ATO-Sol was smaller

17

than that of MSN@ATO for ATO-sol can directly diffuse into cells while

18

MSN@ATO released the drug sustainedly. The IC50 of PAA-MSN@ATO was

19

smaller than MSN@ATO, which maybe resulted from the increased drug release for

20

pH-trigger in cells. After encapsulated by the phospholipid bilayer, the IC50 of

21

LP-PAA-MSN@ATO was reduced for it reduced the aggregation of silica-based drug

22

delivery systems and increased the affinity of drug delivery systems to the cells.

PAA-MSN@ATO,

LP-PAA-MSN@ATO


and

Page 25 of 38 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 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Compared

with

LP-PAA-MSN@ATO,

the

higher

cytotoxicity

of

2

ANG-LP-PAA-MSN@ATO on C6 cells was likely due to the higher concentration of

3

ATO in cells through the targeting effect of angiopep-2. These results indicated that

4

the delivery and uptake of drug-loaded nanoparticles by C6 cells could be enhanced

5

by coating lipid and with the presence of angiopep-2 as targeting molecular,

6

respectively. Thus ANG-LP-PAA-MSN@ATO exhibited the best anti-glioma effect

7

in vitro.

8 9

Figure 6 The viability of HBMEC cell (A) and C6 cell (B) after being treated with NH2-MSN,

10

PAA-MSN, LP-PAA-MSN and ANG-LP-PAA-MSN at concentrations ranging from 0.1 μg/mL to 100

11

μg/mL for 48 h (n=3). The viability of HBMEC (C) and C6 cells (D) after being treated with ATO

12

formulations at a concentration ranged from 1 μM to 20 μM for 48 h (n=3). IC50 values (E) to HBMEC

13

and C6 cells induced by ATO formulations (n=3), *P < 0.05, **P < 0.01 vs HBMEC cells group, #P

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