Superhydrophobic drug-loaded mesoporous silica nanoparticles capped with β-cyclodextrin for ultrasound image-guided combined antivascular and chemo-sonodynamic therapy

Journal Pre-proof Superhydrophobic drug-loaded mesoporous silica nanoparticles capped with βcyclodextrin for ultrasound image-guided combined antivascular and chemosonodynamic therapy Yi-Ju Ho, Cheng-Han Wu, Qiao-feng Jin, Chih-Yu Lin, Pei-Hua Chiang, Nan Wu, Ching-Hsiang Fan, Chia-Min Yang, Chih-Kuang Yeh PII:

S0142-9612(19)30841-5

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119723

Reference:

JBMT 119723

To appear in:

Biomaterials

Received Date: 8 July 2019 Revised Date:

15 November 2019

Accepted Date: 21 December 2019

Please cite this article as: Ho Y-J, Wu C-H, Jin Q-f, Lin C-Y, Chiang P-H, Wu N, Fan C-H, Yang C-M, Yeh C-K, Superhydrophobic drug-loaded mesoporous silica nanoparticles capped with β-cyclodextrin for ultrasound image-guided combined antivascular and chemo-sonodynamic therapy, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2019.119723. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Superhydrophobic Drug-loaded Mesoporous Silica Nanoparticles Capped with

2

β-cyclodextrin for Ultrasound Image-guided Combined Antivascular and

3

Chemo-Sonodynamic Therapy

4 5

Yi-Ju Ho a, Cheng-Han Wu a, Qiao-feng Jin

a, b

6

Nan Wu a, Ching-Hsiang Fan a, Chia-Min Yang c, d, and Chih-Kuang Yeh a,*

, Chih-Yu Lin

c, d

, Pei-Hua Chiang a,

7 8

a

9

Hua University, Hsinchu, Taiwan.

Department of Biomedical Engineering and Environmental Sciences, National Tsing

10

b

11

University of Science and Technology, Wuhan 430022, China

12

c

Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan.

13

d

Frontier Research Center on Fundamental and Applied Sciences of Matters, National

14

Tsing Hua University, Hsinchu, Taiwan.

Department of Ultrasound, Union Hospital, Tongji Medical College, Huazhong

15 16

* Corresponding Authors:

17

Prof. Chih-Kuang Yeh

18

Department of Biomedical Engineering and Environmental Sciences, National Tsing

19

Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu, Taiwan 30013, Tel:

20

+886-3-571-5131 ext. 34240; Fax: +886-3-571-8649.

21

E-mail address: [email protected] (C.-K. Yeh)

22 23

Word count: Abstract 141

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Display items: 5 figures

25

Reference count: 34

26

Supplemental Data: 2 table, 15 figures 1

1

Abstract

2

Interfacial nanobubbles (INBs) on a superhydrophobic surface has been

3

proposed as a solid cavitation agent for enhancing inertial cavitation dose and

4

ultrasound contrast imaging, but the dispersibility of superhydrophobic particles limits

5

the biomedical application. For this study, we designed superhydrophobic mesoporous

6

silica nanoparticles loaded with the anti-tumor drug Doxorubicin (FMSNs-Dox) for

7

tumor therapy. The β-cyclodextrin was used to cap the superhydrophobic surface of

8

FMSNs-Dox to reduce aggregation without inhibiting the accumulation of INBs. The

9

mean size and a contact angle of FMSNs-Dox was 217±58 nm and 129±3°,

10

respectively. The INBs cavitation on the surface of FMSNs-Dox during ultrasound

11

sonication disrupted tumor vessels to allow a large amount of drug penetrating and

12

trapping within tumors. The reduced tumor perfusion, histological reactive oxygen

13

species staining, and tumor inhibition demonstrated that FMSNs-Dox sonication

14

combined anti-vascular, sonodynamic and chemical therapies in a simple platform.

15

Moreover, the repeatability of INB cavitation by single-injection FMSNs-Dox with

16

multiple ultrasound sonication provided intratumoral ultrasound contrast-enhanced

17

imaging from day 1 to 9 (enhancement of 3.84±0.47 dB). Therefore, the

18

characteristics of FMSNs-Dox with slow biodegradation and acoustic-sensitivity

19

presented intratumoral day-scaled lifetime to provide a probability of repeated

20

combination therapy by single-injection.

21 22

Keywords

23

Mesoporous silica nanoparticle, interfacial nanobubbles, ultrasound contrast agent,

24

bubble cavitation, anti-vascular therapy

25 26 2

1

1.

Introduction

2

Superhydrophobicity was originally discovered in nature in the leaves of plants,

3

wings of butterflies, and feathers of birds [1]. The most well-known example is called

4

the Lotus effect, whereby the Lotus leaf allows water droplets to completely roll off

5

the rough surface and carry dust for self-cleaning [2]. Superhydrophobicity is used for

6

surface modification of self-cleaning and waterproofing on clothes, water pipes, and

7

artificial vascular grafts, as well as in the fabrication of multi-functional particles for

8

various applications [3]. Superhydrophobic surfaces include rough surfaces with low

9

surface energy materials, fluorinated polymers, surface-roughing silicones, and

10

surfaces with controlled crystallization of polyethylene [4].

11

Mesoporous silica nanoparticles (MSNs), which possess high drug loading

12

capacity and biocompatibility, have been developed as an efficient drug delivery

13

carrier [3]. In previous studies, the hydrophobic surface on MSNs has been modified

14

to show the accumulation of air nanobubbles at the interface between water and

15

hydrophobic MSNs surface [5, 6]. These interfacial nanobubbles (INBs) are entrapped

16

at the cavities provided by the mesopores of hydrophobic MSNs and are stable for at

17

least 4 days [6, 7]. Yildirim et al. used hydrophobic MSNs to enhance the cavitation

18

and the contrast of ultrasound (US) imaging [8, 9]. Thus, these hydrophobic MSNs

19

have been proposed as nanosized US contrast agents or solid cavitation agents.[10]

20

Since the enhancement of cavitation and image contrast are correlated with the

21

amount of INBs, we previously designed MSNs with superhydrophobicity to improve

22

cavitation by increasing the accumulation of INBs [11]. Bubble cavitation can be

23

separated into the stable and inertial cavitation depending on the intensity of US

24

stimulation. Inertial cavitation occurs during the violent bubble expansion, nucleation,

25

and disruption under high intensity US stimulation [12, 13]. Bubble inertial cavitation

26

not only generates a mechanical force that disrupts the vessels, but also produces 3

1

reactive oxygen species (ROS) that damage the cells [14-16]. During bubble

2

cavitation, ·H and ·OH are produced initially by water pyrolysis inside the cavitated

3

bubbles. The further formation of H2O2, peroxyl radicals, or other ROS could kill

4

tumor cells, which is an acceptable mechanism for sonodynamic therapy [16, 17].

5

Moreover, superhydrophobic modification of the drug-loaded MSNs surface

6

might enable isolation of drugs and aqueous solution via INBs to reduce drug leakage,

7

allowing local release of drugs triggered by INBs cavitation during US stimulation.

8

Since the abnormal tumor vessels have gaps on irregularly arranged endothelial cells,

9

drug-loaded MSNs can passively penetrate and accumulate in the tumor tissue [18-20].

10

In addition, the anti-vascular effect induced by bubble cavitation can increase the drug

11

penetration, which allows drugs to diffuse through the disrupted vessel wall into the

12

tumor tissue [14, 21]. Complete degradation/excretion, a prerequisite for a drug

13

carrier, has been shown to be readily fulfilled by hydrophilic MSNs. However,

14

hydrophobic MSNs could potentially raise a concern due to the repellence against

15

hydrolytic degradation, and the unpredictable agglomerates or aggregates could

16

reduce drug delivery efficacy, leading to unwanted side effects and lowered

17

therapeutic outcomes [22, 23]. Therefore, MSNs should be properly designed to meet

18

both stability within a therapeutic timeframe and subsequent degradability.

19

Herein, we designed multi-functional MSNs, which contain both acoustic

20

sensitive

(superhydrophobic

fluorocarbon)

21

β-cyclodextrin) moieties (Fig. 1). The fabrication starts with covalent attachment of

22

perfluorodecyltriethoxysilane (PFDTS) to the MSNs surface, followed by

23

encapsulation of anti-cancer drug doxorubicin (Dox) into MSNs mesopores. PFDTS

24

is a fluoroalkylsilane with a chemical formula of F3C(CF2)7(CH2)2-Si(OC2H5)3

25

featuring low surface energy and superhydrophobicity due to its fluoridated carbons.

26

It has been used as a superhydrophobic agent for coatings [24, 25] and was used in 4

and

stabilizing

(amphiphilic

1

our previous studies for MSNs superhydrophobization [11, 26]. Then, enlightened by

2

previous work, we capped the external surface of the FMSN-Dox with β-cyclodextrin

3

(β-CD) with an internal hydrophobic cavity and external hydrophilic groups to

4

improve dispersibility without attenuating the accumulation of the INBs [27]. The

5

material, designated as FMSNs-Dox, possessed good dispersity in aqueous solution,

6

sufficient drug loading, adequate resistance against hydrolytic degradation within a

7

therapeutic timeframe, and acoustic sensitivity. In addition, premature Dox release

8

can be significantly mitigated by the fluorocarbon chain. The INB cavitation produced

9

by FMSNs-Dox was evaluated based on the efficiency of drug release, inertial

10

cavitation dose (ICD), and yield of ROS to demonstrate the combination of chemical,

11

physical, and sonodynamic therapy. We traced the in vivo contrast enhancement of US

12

imaging, tumor growth, and mouse survival after single-dose FMSNs-Dox treatment

13

to establish that INBs could exist within tumors for days and provide repeatable

14

cavitation for continuous tumor therapy. Moreover, the stable yet steadily degrading

15

FMSNs-Dox could release drugs in a slow and continuous fashion over a relatively

16

long period of time to target regions, which might provide the opportunity to regulate

17

the treatment protocols and reduce side effects.

18

5

1 2

Fig. 1. The superhydrophobic surface of FMSNs-Dox accumulates INBs to

3

induce bubble cavitaiton, ROS generation, and drug release during US

4

stimulation. After single-injection FMSNs-Dox, the anti-vascular effect is

5

accomplished by INB cavitation to increase the intratumoral accumulation of

6

FMSNs-Dox. The low biodegradation rate of FMSNs-Dox prolonges the in vivo

7

persistence of INBs, and the intratumoral INBs could be repeatably cavitated to

8

improve sonodynamic and chemical therapies under multiple US sonications.

9

The repeatability of INB cavitation provides a platform to combine anti-vascular,

10

sonodynamic, and chemical therapies by single-dose FMSNs-Dox treatment.

11 12

2.

Materials and methods

13

2.1. Materials 6

1

Benzylcetyldimethylammonium chloride (BCDAC), diethylene glycol hexadecyl

2

ether

(C16E2),

cetyltrimethylammonium

bromide

3

1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine

4

dihydroethidium (DHE) were purchased from Sigma Aldrich (St Louis, USA).

5

Tetraethoxysilane (TEOS) was obtained from Acros (Belgium, USA). Doxorubicin

6

(Dox) hydrochloride was obtained from Seedchem Company PTY.LTD (Melbourne,

7

Australia). Hydrogen chloride (37%, w/w) and ammonia (28%) were purchased from

8

Showa (Tokyo, Japan). Perfluorodecyltriethoxysilane (PFDTS) and β-cyclodextrin

9

(β-CD) were purchased from Alfa Aesar (Ward Hill, USA). All the solvents and

10

reagents were analytical reagents. All the aqueous solutions were prepared using

11

deionized Millipore Milli-Q water. For the in vitro cellular level experiments,

12

Dulbeco's modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and

13

penicillin–streptomycin (PS) were obtained from Gibco (Grand Island, USA). For

14

histological immunostaining, rat-anti-mouse CD31, and FITC-conjugated goat anti-rat

15

IgG were purchased from GeneTex (Irvine, USA).

perchlorate

(CTAB), (DiI),

and

16 17

2.2. Particle synthesis and functionalization

18

Briefly, 0.74 g of BCDAC, 0.26 g of C16E2, 21.4 mL of 0.4 M NaOH, and 575

19

mL of water were added to a polyethylene bottle and stirred at 35 °C overnight. TEOS

20

(5.98 mL) was then injected at a rate of 7.5 mL per hour, followed by aging at 90 °C

21

for 24 h. The MSNs were filtrated, then washed with water and acetone, and dried

22

under ambient conditions. The MSNs (0.1 g) was heated at 150 °C in vacuum for 12 h

23

to remove the adsorbed water and then dispersed in a solution containing 1 mL of

24

PFDTS and 10 mL of toluene [28]. The mixture was stirred at 100 °C for 48 h, and

25

the product was collected by filtration, and washed with ethanol. Finally, the FMSNs 7

1

were obtained after removing surfactants by repeated ion exchange in a dilute

2

HCl-ethanol solution (2% in v/v) at 35 °C and dried at 60 °C for 12 h.

3 4

2.3. Characterization of FMSNs

5

Nitrogen physisorption isotherms were measured at 77 K using a Quantachrome

6

Autosorb-1MP instrument. The pore volumes were evaluated at a relative pressure of

7

0.95, and the adsorption branches in the relative pressure range of 0.05–0.30 were

8

used to calculate surface areas by applying the Brunauer-Emmett-Teller method.

9

X-ray diffraction patterns were recorded on a Mac Science 18MPX diffractometer

10

using Cu Kα radiation. Thermogravimetric analysis (TGA) was carried out in air using

11

Mettler-Toledo, 2-HT. Transmission electron microscopy (TEM) images were obtain

12

using a JEOL JEM-2010 microscope operating at 200 kV. The size distributions and

13

ζ-potentials of the nanoparticles were measured using dynamic light scattering

14

(Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). All of the samples

15

were diluted with 18.2-MΩ milipore MiliQ water.

16 17

2.4. Drug loading and β-CD capping

18

Subsequently, the anti-cancer drug Dox was loaded onto the FMSNs under the

19

assist of ethanol. Briefly, 15 mg Dox and 10 mg FMSNs were dissolved in 1 mL 75%

20

of ethanol solution. The ethanol was vaporized at 60 °C, and the process was repeated

21

3 times by replenishing anhydrous ethanol when the ethanol was almost dried. Finally,

22

the Dox-loaded FMSNs (denoted as FMSNs-Dox) were centrifuged at 17500 g for 5

23

min and washed with deionized water 3 times to remove the free Dox. The obtained

24

FMSNs-Dox were further dried for 48 h in a drying oven and stored at 4 °C for the

25

following experiments. FMSNs-DiI were prepared with the protocol as described 8

1

above by exchanging Dox with DiI (0.625 mg/mL). Finally, FMSNs-Dox were

2

dispersed in various concentrations of β-CD and incubated for 24 h, and then

3

collected by centrifugation at 15000 g for 5 min. After capping β-CD, FMSNs-Dox

4

were re-dispersed in 10 mM phosphate-buffered saline (PBS) and stored at 4 °C. All

5

samples were capped with β-CD in the in vitro and in vivo experiments. To evaluate

6

the stability of drug loading on FMSN-Dox, samples (0.1 mg/mL) were maintained at

7

37 °C water bath and then centrifuged at 17500 g for 5 min to collect the free Dox at

8

various time points. The Dox leakage was determined by a plate reader system (Tecan

9

Infinite M200, Tecan Trading AG, Switzerland) at an absorbance wavelength of 490

10

nm and normalized to the zero time point.

11 12

2.5. ICD detection during INBs cavitation

13

Since bubble inertial cavitation occurred during violent bubble expansion, the

14

detection of ICD might provide evidence to support the existence of INBs on the

15

FMSNs-Dox surface. The ICD was measured with passive cavitation methods as

16

described in our previous study [29]. Samples were diluted to 0.1 mg/mL in deionized

17

water (DIW) and infused into PE50 tubing (Micro-Renathane, Braintree Scientific,

18

Braintree, USA) at the flow rate of 0.1 mL/min by syringe pump (KDS120, KD

19

Scientific, New Hope, USA). A 2-MHz high intensity-focused ultrasound (HIFU) was

20

driven by a power amplifier (150A100B, Amplifier Research, Souderton, PA, USA)

21

and a waveform generator to transmit 50-cycle pulses with various peak-negative

22

acoustic pressures and pulse repetition frequency (PRF) of 100 Hz for stimulating

23

FMSNs-Dox (0.1 mg/mL). A 15-MHz focused transducer (V303, Olympus, Waltham,

24

MA, USA) was used to receive broadband emission signals. The IC signals were

25

received using an oscilloscope (LT322, LeCory, Chestnut Ridge, NY, USA). The 9

1

recorded time-domain signal was first transformed into a frequency domain spectrum

2

using fast Fourier transform. The area under the frequency spectrum from 10 to

3

20 MHz were termed ICD and used to assess the IC capability and intensity. Since the

4

β-CD could improve the dispersibility of FMSNs-Dox, the size distribution of

5

FMSNs-Dox was measured to evaluate the separation of β-CD from FMSNs-Dox

6

after US sonication.

7

To observe the cavitation of INBs during US stimulation, a high-speed camera

8

(FASTCAM SA4, Photron, Tokyo, Japan) was mounted on an inverted microscope

9

(IX71, Olympus, Tokyo, Japan) with a 63× objective (Fig. S1). Diluted FMSNs-Dox

10

(0.1 mg/mL) were injected into a 200 µm cellulose tube (Spectrum Labs, Rancho

11

Dominguez, CA, USA) at a flow rate of 1 mL/h by syringe pump. A 2-MHz HIFU

12

transducer was focused on the 200 µm cellulose tube to stimulate FMSNs-Dox by

13

3-cycle single pulse. The short pulse was used to reduce the motion of cellulose tube

14

after US stimulation.

15 16

2.6. ROS detection

17

For the in vitro ROS measurement, the 2',7'-dichlorofluorescein-diacetate

18

(DCFH-DA) dye was first hydrolyzed with 0.01 M sodium hydroxide and diluted by

19

0.1 M PBS. We used FMSNs in this experiment because the fluorescence of

20

FMSNs-Dox might interfere with the detection of ROS by DCFH-DA. FMSNs at a

21

concentration of 0.1 mg/mL and 10 µM DCFH-DA were injected into the

22

polyethylene tube at a velocity of 0.1 mL/min using a syringe pump. The US

23

parameters for the in vitro (6 MPa, 50-cycle, PRF of 100 Hz, 5 min) and in vivo (6

24

MPa, 5000-cycle, PRF of 18 Hz, 10 min) experiments were used to evaluate the

25

generation of ROS for sonodynamic therapy. A longer cycle number for US was used 10

1

for the in vivo experiments to increase the penetration of US within solid tumors. The

2

PRF was regulated following the flow rate of FMSNs-Dox to establish that all the

3

FMSNs-Dox were stimulated during US sonication. The fluorescence intensity was

4

measured using a plate reader (Infinite 200, Tecan Group, Mannedorf, Zurich,

5

Switzerland) at excitation 480 nm and emission 530 nm.

6 7

2.7. Cell Viability

8

The transgenic adenocarcinoma mouse prostate (TRAMP) cell line was used to

9

evaluate the cell viability of FMSNs-Dox treatment. The TRAMP cells were cultured

10

in DMEM supplemented with 10% FBS and 1% PS under a humidified atmosphere

11

with 5% CO2 at 37 °C for in vitro experiments. The cells were subcultured in a

12

96-well plate (105 cells/well) and incubated for 24 h. The experimental groups

13

included cell, Dox, MSNs, MSN-Dox, FMSNs, and FMSNs-Dox, each group was

14

further separated into without and with US groups. The cell group was the control

15

group, that was not added samples. The mounts of Dox in the MSNs-Dox and

16

FMSNs-Dox groups were adjusted to have an equivalent Dox concentration in the

17

Dox group (LD50=10 µg). Cell layers were sonicated with 5 MPa and 50-cycle pulses

18

at PRF of 100 Hz for 5 min. After 4 h, cells were washed with PBS and replaced with

19

fresh medium for 24 h culture. Finally, cell viability was measured using the

20

almarBlue assay (BUF012B, AbD Serotec, Kidlington, UK). MSNs were used to

21

determine that FMSNs could disrupt cells via INBs cavitation due to the

22

superhydrophobic surface. The comparison between Dox, MSN-Dox, and FMSN-Dox

23

was designed to demonstrate that drug carriers could reduce cytotoxicity to protect

24

normal tissue. The FMSNs-Dox with US group presented the cytotoxicity under the

25

combination of chemical, physical, and sonodynamic therapy.

26 11

1

2.8. In vivo anti-vascular effect

2

The in vivo anti-vascular effect was evaluated by the window chamber mouse

3

model (Fig. S2) [30]. C57BL/6JNarl mice (age: 7–10 weeks; weight: 30–35 g) were

4

purchased from the National Laboratory Animal Center (Taipei, Taiwan). All the

5

animal experiments were approved by the animal experiment committee at National

6

Tsing Hua University (approval number: 107027) following the guidelines of the

7

Institutional Animal Care Committee. For the experimental setup, the window

8

chamber model was mounted on mouse dorsal skin to observe vascular changes under

9

sonication via the cover glass window in window chamber kits (SM100, APJ Trading,

10

Ventura, CA, USA) under the acoustic-optical system [31, 32]. Mice (N=3) received

11

100 µL FMSNs-DiI (1 mg/mL) via retro-orbital injection. FMSNs-DiI provided good

12

intravital fluorescent tracing that was used to simulate the in vivo distribution of

13

FMSNs-Dox. The 2-MHz HIFU transducer was manually triggered to transmit a

14

5000-cycle single pulse at a desired time point with different pressures.

15 16

2.9. Tumor sonication

17

For the solid tumor model, TRAMP cells (1×106 cells) were subcutaneously

18

implanted into the right legs of mice and allowed to grow for 7 days (tumor diameter

19

of 6 mm). After administration of 100 µL FMSNs-Dox (1 mg/mL, 150 µg Dox/mouse)

20

into mice via retro-orbital injection, tumors were sonicated under a 2-MHz HIFU

21

scanning using a triaxial platform at 1 mm intervals [30]. The parameters of the HIFU

22

sonication were employed with a peak negative pressure of 6 MPa, 5000-cycle, and

23

PRF of 18 Hz. In total, the sonication time was about 20-30 min. The commercial

24

7-MHz US imaging system (model t3000, Terason, Burlington, USA) was used to

25

provide imaging guidance during tumor sonication. 12

1 2

2.10.

Tumor perfusion tracing

3

Information on tumor perfusion due to microbubble infusion was acquired to

4

evaluate the difference between pre- and post-treatment by US B-mode imaging. The

5

homemade microbubbles with a mean size of 0.2-0.7 µm were at a concentration of

6

1.4-3.0×1010 microbubbles/mL; the fabrication details were described in our previous

7

studies [14]. The imaging dosage was 1×107 microbubbles per mouse by retro-orbital

8

injection. Before treatment, the initial images showed the tumor contour, and then the

9

perfusion images were recorded to define the baseline images after the first injection

10

of microbubbles. Solid tumor treatment with FMSNs-Dox injection was started after

11

microbubble injection of 30 min to avoid microbubble cavitation during HIFU

12

sonication. After treatment, tumor perfusion images were acquired at 1, 30, and 60

13

min by multiple microbubble injections to evaluate the anti-vascular effect via

14

perfusion recovery. The gray level intensity of B-mode images within the tumor

15

region was measured to calculate the percentage of perfusion intensity relative to the

16

baseline images.

17 18

2.11.

Histological assessments

19

Tumors were removed 24 h after treatment for histological assessment. Tissue

20

sections (thickness: 20 µm) were stained with hematoxylin and eosin (H&E) and

21

CD31 to indicate tissue damage and vascular pattern, respectively. The intratumoral

22

ROS and drug distributions were evaluated using FMSNs and FMSNs-DiI,

23

respectively, to avoid the fluorescent interference between hemorrhage, Dox, and dye.

24

The dihydroethidium (DHE) dye was used to evaluate the endogenous ROS levels

25

with red fluorescence. The tumor histological images were captured to analyze the FI 13

1

of ROS and FMSNs-DiI.

2 3

2.12.

Biodistribution and biodegradability of FMSNs-DiI

4

After demonstrating the anti-vascular, sonodynamic, and chemical effects of

5

FMSNs-Dox sonication, the biodistribution of superhydrophobic nanoparticles was

6

further investigated using FMSNs-DiI. After 24 and 48 h of FMSNs-DiI injection,

7

mice liver, spleen, lung, kidney, heart, and tumor were removed to record ex vivo

8

fluorescence imaging by an in vivo imaging system (IVIS Spectrum, Caliper Life

9

Science, Hopkinton, USA).

10

In order to investigate the intratumoral lifetime of FMSNs-Dox, the in vitro

11

biodegradability of FMSNs-Dox was estimated. FMSNs-Dox were cultured in the

12

simulated body fluid at 37 °C to simulate the in vivo environment. The simulated

13

body fluid is a buffered solution (pH 7.4) which is used to simulate the ionic

14

compositions and strength of human blood plasma to investigate the in vivo behavior

15

and release mechanisms of particles [33]. The TEM images and ICD detection were

16

estimated at various culture time points to evaluate the structure of FMSNs-Dox and

17

the ability of INBs cavitation, respectively.

18

Moreover, the long-term potential toxicology of FMSNs-Dox was evaluated by

19

blood analysis and histological assessments. Tumor-bearing mice were intravenously

20

injected FMSNs-Dox (150 µg Dox/kg) at day 1, and then collected blood from eye

21

socket at day 2, 5, and 10. The whole blood samples were analyzed by AmiShieldTM

22

(Protectlife, Taoyuan, Taiwan). For histological assessments, liver, kidney, and lung

23

were removed after 48 h FMSNs-Dox injection. Tissue sections (thickness: 20 µm)

24

were stained with H&E to indicate long-term potential damage for organs.

25 14

1

2.13.

Tumor therapy by single-injection FMSNs-Dox

2

The efficacy of tumor therapy by FMSNs-Dox was evaluated in a single

3

injection. The initial tumor volume was 69±17 mm3 on day 7 after tumor cell

4

implantation. Tumor-bearing mice were intravenously injected with FMSNs-Dox

5

(150 µg Dox/kg) at day 1, and then tumors were sonicated at day 1, 3, 5, and 7 (Fig.

6

S3). Since FMSNs-Dox might accumulate and maintain structure in tumors, the in

7

vivo repeatability of INBs cavitation from FMSNs-Dox was assessed by US contrast

8

enhancement imaging during tumor sonication. Finally, the mouse survival after

9

treatment was monitored and analyzed by Prism 6 software (GraphPad Co., San

10

Diego, CA, USA).

11 12

2.14.

Statistical evaluation

13

All data are shown as mean and standard deviation with more than three

14

independent samples, and the standard deviation is shown as an error bar in each

15

graph. The paired two-tailed Student’s t-test was used to establish the statistically

16

significant difference between two individual groups when p value < 0.05. Mouse

17

survival curves were generated using the Kaplan–Meier method.

18 19

3.

Results and discussion

20

3.1. Superhydrophobic, drug-loaded, dispersible nanoparticles

21

The parent MSNs with Ia3d mesopore symmetries were synthesized via the

22

sol-gel method, and the MSNs were conjugated with PFDTS for superhydrophobicity.

23

The successful fluoridation of FMSNs was confirmed by TGA, with a demonstrated

24

weight loss of ~48 % from the fluorocarbon (Fig. S4). Powder X-ray diffraction

25

patterns showed that the well-ordered mesostructure was maintained after fluoridation 15

1

(Fig. 2A). F-MSNs feature type IV nitrogen physisorption isotherm (Fig. 2B), from

2

which the derived surface area and pore volume were 216 m2/g and 0.14 cm3/g,

3

respectively.

4

5 6

Fig. 2. Characterization of synthesized FMSNs-Dox. (A) Powder X-ray

7

diffraction patterns.. (B) Nitrogen physisorption isotherms. (C) Dox loading

8

amount and efficiency. (D) Dox leakage. (E) Mean size and contact angle of

9

FMSNs with various concentrations of β-CD. (F) TEM and contact angle images.

10

(* p<0.05; ** p<0.01)

11 12

The optimal fabrication of FMSNs-Dox was determined at the weight ratio

13

(Dox/FMSNs) of 1.5, which showed the Dox loading amount of 1.40±0.06 mg and 16

1

the efficiency of 93.2±0.4% (Fig. 2C). Effective loading of Dox onto FMSNs was

2

evidenced by the nitrogen physisorption analysis shown in Fig. 2B. The pore volume

3

of F-MSNs was almost completely taken up by Dox after repetitive loading, and the

4

derived surface area and pore volume of FMSNs-Dox were 35 m2/g and 0.04 cm3/g,

5

respectively. FMSNs-Dox possessed 13.4±1.2% leakage at 37 °C after 48 h, which

6

was noticeably lower than that of the non-fluoridated counterpart MSNs-Dox,

7

allowing the potential for low drug release (Fig. 2D). Finally, FMSNs-Dox were

8

dispersed in various concentrations of β-CD, the mean sizes and contract angles of

9

FMSNs decreased as the β-CD concentration increased (Fig. 2E). Since

10

superhydrophobicity is determined by a water contact angle on the material surface

11

close to or higher than 150°, the optimal β-CD concentration was 8 mg/mL, in which

12

FMSNs-Dox of a mean size of 217±58 nm and a contact angle of 129±3° were

13

obtained. The reduced contrast between mesopore and silica wall under TEM

14

observation once again indicated the existence of organic species, and the variation in

15

surface hydrophobicity can be visualized by the contact angle images (Fig. 2F and S5).

16

The size, zeta-potentials, and contact angle of FMSNs-Dox are summarized in Table

17

S1. All the samples were capped with β-CD in the in vitro and in vivo experiments

18

described below. The FMSNs agglomeration after Dox loading should be attributed to

19

reduced surface potential. Since Dox molecule exists in cationic form in biological

20

and acidic environments, the negative charge of FMSNs would be partly neutralized.

21

In addition, the high concentration of Dox would aggregate together to increase the

22

size of FMSNs-Dox. After FMSNs-Dox capping with β-CD, the size was reduced due

23

to the improving dispersibility of FMSNs-Dox.

24 25

3.2. INBs cavitation on FMSNs-Dox 17

1

For the evaluation of INBs cavitation capabilities on the FMSNs-Dox surface, the

2

in vitro ICD, high-speed imaging, ROS, cell viability, and anti-vascular effect were

3

investigated. In Fig. 3A, the ICD of hydrophilic MSNs showed non-significant

4

differences when compared to DIW at each acoustic pressure. Although Dox loading

5

and β-CD capping slightly reduced the superhydrophobicity of FMSNs-Dox (Fig. S6),

6

the ICD induced by FMSNs-Dox was significantly higher than that of MSNs. The size

7

distribution of FMSNs-Dox showed no significant difference between the without

8

(w/o) and with (w/) US sonication groups (Fig. S7). This result indicated that β-CD

9

would not be separated from FMSNs-Dox after US sonication.

10

11 18

1

Fig. 3. INBs cavitation. (A) ICD detection. (B) High-speed images and the mean

2

size of visualized bubbles during INBs cavitation. (C) ROS detection. (D) Cell

3

viability. The legends of w/o and w/ mean without US and with US sonication,

4

respectively. (E) Intravital images for anti-vascular effect evaluation. (F)

5

Intertissue FI of FMSNs-DiI after vascular disruption. (* p<0.05; ** p<0.01)

6 7

High-speed images (100k fps) displayed micro-sized bubble cavitation from

8

FMSNs-Dox during US sonication, demonstrating the presence of INBs cavitation.

9

(Fig. 3B). The diameters of the visual bubbles were 30±3, 34±5, and 40±5 µm at

10

acoustic pressures of 4, 5, and 6 MPa, which were much larger than the original size

11

of the FMSNs-Dox. During INBs cavitation, INBs expanded and coalesced with

12

neighboring INBs to form micro-sized bubbles [34, 35]. Water adjacent to the

13

superhydrophobic surface forms a thin layer with a low density that might be

14

vaporized by the heat produced by inertial cavitation to increase the volume of INBs

15

[6, 36]. The detection of ROS generated by FMSNs sonication showed enhancement

16

that was more significant than that by MSNs sonication (Fig. 3C). Comparison of the

17

in vitro and in vivo US parameters revealed that high duty US could initiate inertial

18

cavitation and generate about 12-fold more ROS.

19 20

3.3. Cytotoxicity of FMSNs-Dox sonication

21

The cell viability of FMSNs-Dox sonication under various acoustic pressures is

22

presented in Fig. S8. The cell viability was reduced to 68±7, 41±5, and 44±6% under

23

acoustic pressure of 4, 5, and 6 MPa, respectively. There was no significant difference

24

between the acoustic pressure of 5 and 6 MPa. In the principle of as low as reasonably

25

achievable, our study used acoustic pressure to 5 MPa for the in vitro cell experiments 19

1

to investigate the contribution from chemical, physical, and sonodynamic therapy. In

2

Fig. 3D, the bioeffects induced by inertial cavitation showed 57±3% by FMSNs

3

sonication. The cell viability was maintained at 79±6% in the FMSNs-Dox group, and

4

was then reduced to 41±5% after sonication. These results indicate that FMSNs-Dox

5

produced less cytotoxicity from drug leakage and was responsive to US, resulting in

6

disruption of tumor cells by INBs cavitation.

7 8

3.4. Anti-vascular effect induced by INBs cavitation

9

In order to evaluate the in vivo circulation time of FMSNs, the intravital

10

fluorescent images were recorded without US stimulation to analyze the intravascular

11

fluorescent intensity (FI) over time (Fig. S9). The relative FI at 150 min showed no

12

significant difference to the pre-injection images. The in vivo circulation time of

13

FMSNs-DiI was less than 150 min. The intravital images revealed vascular disruption,

14

hemorrhage, and FMSNs-DiI penetration after US sonication (Fig. 3E). The

15

percentage of FI in tissue was proportional to the acoustic pressure and increased over

16

time (Fig. 3F). We further analyzed the anti-vascular effect at different vessel sizes

17

and found that the proportion of vascular disruption was 100% at vessel size under 10

18

µm (Fig. S10). The vessel size between 30 to 40 µm presented 57% vascular

19

disruption under 6 MPa. Interestingly, the size of the blood vessel disruption agreed

20

well with the size of the cavitated INBs observed in the high-speed imaging.

21

According to these results, 6 MPa US pulses were applied in subsequent tumor

22

treatments. Thus, the INBs cavitation induced by FMSNs-Dox sonication could

23

generate mechanical force, ROS, and slow release Dox to provide a platform for

24

combining anti-vascular, sonodynamic, and chemical therapies.

25 20

1

3.5. Intratumoral bioeffects from FMSNs-Dox sonication

2

The in vivo bioeffects of INBs cavitation were further investigated in

3

tumor-bearing mice. The efficiency of anti-vascular effect induced by FMSNs-Dox

4

sonication was evaluated by the perfusion recovery after treatment. Fig. 4A reveals a

5

significant reduction in tumor perfusion after sonication and shows slight perfusion

6

recovery at 60 min. The quantification of tumor perfusion intensity was 50±8, 53±5,

7

and 50±8% at 1, 30, 60 min, respectively, and the perfusion was no statistically

8

significant recovery, demonstrating the efficient anti-vascular effect (Fig. 4B).

9

10 11

Fig. 4. Bioeffects of FMSNs-Dox sonication. (A) Tumor perfusion imaging for

12

estimating anti-vascualr therapy. (B) Quantificantion of tumor perfusion. (C)

13

Histological images of H&E and CD31 staining to present tissue necrosis and 21

1

vessel pattern, respectively. (D) Intratumoral distribution of ROS and

2

FMSNs-DiI accumulation. (** p<0.01)

3 4

Histological assessment was used to evaluate the intratumoral vascular disruption,

5

ROS generation, and drug penetration by FMSNs-Dox sonication after 24 h.

6

Histological images with H&E staining showed the tissue necrosis and hemorrhage

7

induced by INBs cavitation in the FMSNs+US and FMSNs-Dox+US groups (Fig. 4C).

8

Tumor vessels revealed a hazy pattern in CD31 staining to indicate the occurrence of

9

vascular disruption, which agreed well with the results obtained in tumor perfusion

10

imaging. FMSNs and FMSNs-DiI were used to evaluate the distribution of ROS and

11

drugs, respectively, within tumors to avoid the fluorescent interference between

12

hemorrhage, ROS detector DHE, and Dox. The intratumoral fluorescent distribution

13

indicated significant enhancement of ROS after US sonication to demonstrate the

14

potential use of FMSNs-Dox for sonodynamic therapy (Fig. 4D). Moreover, the

15

accumulation of FMSNs-DiI increased 2.98-fold in the FMSNs-DiI+US group

16

relative to the FMSNs-DiI group since vascular disruption could improve drug

17

penetration within tumors.

18 19

3.6. Repeatability of INBs cavitation within tumors

20

The biodistribution of superhydrophobic nanoparticles showed accumulation of

21

FMSNs-DiI in livers after 24 h injection, but rapid decay at 48 h (Fig. 5A and S11).

22

Although the FMSNs-DiI accumulation in tumors at 24 h was less than that of livers,

23

the radiant efficiency of FMSNs-DiI within tumors showed non-significant decay and

24

was maintained for 48 h.

25 22

1 2

Fig. 5. Repeatability of INBs cavitation. (A) Biodistribution of FMSNs-DiI

3

without US sonication. (B) ICD detection of FMSNs-Dox at various culture times.

4

FMSNs-Dox were cultured in the simulated body fluid at 37 °C. The evaluation

5

of tumor treatment outcome was traced after single injection of FMSNs-Dox with

6

multiple US sonication. (C) US contrast enhancement imaging induced by INBs

7

cavitation. (D) Quantification of US enhancement signals. (E) Relative tumor

8

volume tracing. (F) Mouse survival curve. (* p<0.05; ** p<0.01)

9 10

The in vitro biodegradability of FMSNs-Dox showed partial degradation at the

11

center of FMSNs-Dox after 7 days culture in the TEM images (Fig. S12). The

12

degradation of MSNs is via hydrolytic dissolution of Si-O-Si bonds in silica 23

1

framework [37]. For FMSNs, the superhydrophobic fluorocarbon groups are located

2

on and near the external surface, where the rate of hydrolysis of Si-O-Si bonds is

3

largely reduced. This leads to much slower silica dissolution near hydrophobic

4

external surface than hydrophilic internal surface of FMSNs, therefore the particles

5

mainly degrade from the inside. Although the structure of FMSNs-Dox might be

6

destroyed by TEM, the visualized difference between day 0 and 7 still revealed

7

evidence of repellence against hydrolytic degradation of FMSNs-Dox. The ability of

8

INBs cavitation was also evaluated by ICD detection at various culture time points.

9

Although ICD was inversely proportional to the culture time, FMSNs-Dox still

10

presented much higher ICD than simulated body fluid only at day 14 (Fig. 5B). In

11

TEM images, the structure of FMSNs-Dox revealed a non-visualized difference after

12

US sonication to indicate that US sonication would not disrupt FMSNs-Dox (Fig.

13

S13). These above results demonstrate the potential for using repeatable INBs

14

cavitation within tumors after single injection of FMSNs-Dox.

15

The long-term potential toxicology of FMSNs-Dox was also evaluated by blood

16

analysis and organ histological images. Table S2 showed whole blood analysis. The

17

normal group was mice without tumors. Comparison with control group, FMSNs-Dox

18

group showed no significant difference in all the analyzed results at each time point.

19

The histological images revealed no visualized damage in the liver and kidney, but

20

showed edema and inflammatory in the lung due to the pulmonary embolism by

21

FMSNs-Dox (Fig. S14). Therefore, although FMSNs-Dox might induce the partial

22

pulmonary embolism, the long-term potential toxicology of FMSNs-Dox does not

23

influence the function of normal tissue and organs.

24 25

3.7. Treatment outcome by single-injection FMSNs-Dox 24

1

The US images demonstrated that INBs could repeatably exist and cavitate to

2

provide obvious contrast enhancement within tumors from day 1 to day 9 (Fig. 5C).

3

The quantification results showed consistent contrast enhancement of 3.84±0.47 dB

4

from INBs in US imaging from day 1 to 9, which decreased over time (Fig. 5D). After

5

single-injection FMSNs-Dox with multiple US sonications, the relative tumor volume

6

tracing showed gradual reduction in tumor growth in the FMSNs-Dox group to

7

demonstrate slow but continuous drug release from FMSNs-Dox (Fig. 5E). The

8

treatment protocol of the FMSNs+US group further demonstrates the ability of

9

intratumoral repeatable INBs cavitation to restrict tumor growth by combining

10

anti-vascular and sonodynamic therapy. Finally, the combination of anti-vascular,

11

sonodynamic, and chemical therapy in the FMSNs-Dox+US group revealed

12

significant inhibition of tumor growth after single-injection of FMSNs-Dox with

13

multiple US sonication. The body weight of the tumor-bearing mice increased linearly

14

with age and showed no significant difference between groups (Fig. S15). The

15

survival curves showed a maximum survival of 28, 33, 36, and 45 days for control,

16

FMSNs-Dox, FMSNs+US, and FMSNs-Dox+US groups, respectively (Fig. 5F).

17

Therefore, the single injection of FMSNs-Dox with multiple US sonication provided

18

efficient tumor growth inhibition to prolong mouse survival by combining

19

anti-vascular, sonodynamic, and chemical therapy.

20 21

4.

Conclusions

22

In this study, we confirmed that superhydrophobic mesoporous silica drug carrier

23

FMSNs-Dox is multifunctional, providing drug loading, water-dispersity, slow

24

biodegradation, and acoustic-sensitivity. The superhydrophobic modification of

25

FMSNs-Dox prevented drug leakage and increased the accumulation of INBs for 25

1

enhancing efficiency of cavitation. The applications of INBs on the surface of

2

FMSNs-Dox provided bubble cavitation under US sonication to enhance US contrast

3

imaging, disrupt tumor vessels/cells, and generate ROS. The anti-vascular effect

4

induced by FMSNs-Dox sonication disrupted vessels at the maximal size of 40 µm to

5

increase drug penetration, and then inhibited almost 60% of tumor perfusion to starve

6

tumor cells. In addition, the low biodegradation rate of FMSNs-Dox prolonged the in

7

vivo persistence of INBs, and the intratumoral INBs could be repeatably cavitated to

8

improve sonodynamic therapy under multiple US sonications. Hence, our study

9

demonstrates that FMSNs-Dox sonication combines anti-vascular, sonodynamic and

10

chemical therapies in a simple platform. The repeatability of INB cavitation was

11

demonstrated by single-injection FMSNs-Dox with multiple US sonications, which

12

provided in vivo day-scaled US contrast imaging and tumor growth inhibition. Finally,

13

the characteristics of FMSNs-Dox make it an excellent candidate for a modified drug

14

carrier for regulated treatment protocols and reduced side effects in tumor therapy.

15 16 17

Acknowledgements The authors gratefully acknowledge the support of the Ministry of Science and

18

Technology,

Taiwan

under

Grant

No.

MOST

19

107-2627-M-007-005,

20

106-2113-M-007-025-MY3, and 107-3017-F-007-002. This study was also supported

21

by Chang Gung Memorial Hospital (Linkou, Taiwan) under Grant No. 107Q2511E1.

22

The Frontier Research Center on Fundamental and Applied Sciences of Matters from

23

The Featured Areas Research Center Program within the framework of the Higher

24

Education Sprout Project by the Ministry of Education (MOE) in Taiwan also

25

supported our study. Moreover, this study was also supported by National Natural

26

Science Foundation of China under Grant No. 81801716.

107-2119-M-182-001,

26

107-2221-E-007-002,

106-2218-E-007-022-MY3,

1 2

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: