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Multifunctional Peptide-Amphiphile End-Capped Mesoporous Silica Nanoparticles for Tumor Targeting Drug Delivery Yinjia Cheng, Ai-Qing Zhang, Jing-Jing Hu, Feng He, Xuan Zeng, and Xian-Zheng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12647 • Publication Date (Web): 29 Dec 2016 Downloaded from http://pubs.acs.org on January 2, 2017

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ACS Applied Materials & Interfaces

1

Multifunctional Peptide-Amphiphile

2

End-Capped Mesoporous Silica Nanoparticles

3

for Tumor Targeting Drug Delivery

4

5

Yin-Jia Cheng,1,2,* Ai-Qing Zhang,1 Jing-Jing Hu,2 Feng He,2 Xuan Zeng,2,*

6

Xian-Zheng Zhang2

7 1

8

Nationalities, Wuhan 430074, China

9 10

School of Chemistry and Materials Science, South-Central University for

2

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, China

11 12 13 14 15 16 17 18 19

* To whom correspondence should be addressed.

20

E-mail addresses: [email protected] (Y. J. C.), [email protected] (X.

21

Z.)

22 1

ACS Paragon Plus Environment


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1

ABSTRACT: A tumor targeting redox-responsive drug delivery system (DDS)

2

with bioactive surface was constructed by immobilizing peptide-based amphiphile

3

C12-CGRKKRRQRRRPPQRGDS (defined as ADDA-TCPP) onto the mesoporous

4

silica nanoparticles (MSNs) as an end-capping nanovalve, which consist of two main

5

segments: a hydrophobic alkyl chain ADDA and a hydrophilic amino acid sequence

6

containing a Tat48-60 peptide sequence with a thiol terminal group and an RGDS

7

targeting ligand, via a disulfide linkage for redox-triggered intracellular drug delivery.

8

A series of characterizations confirmed that the nanosystem had been successfully

9

fabricated. The anti-tumor drug Doxorubicin (DOX) was selected as a model drug and

10

efficiently trapped in the pores of MSNs, and in vitro release experiment

11

demonstrated

12

(DOX@MSN-ss-ADDA-TCPP)

13

self-assemblies through hydrophobic interactions between the alkyl chains, and the

14

resulting DDS exhibited “zero premature release” of DOX in physical environment.

15

However, a burst drug release was triggered by a high concentration of glutathione

16

(GSH) in simulated cellular cytosol. Moreover, detailed investigations confirmed that

17

incorporation of RGDS peptide facilitated the active targeting delivery of DOX to

18

αvβ3 integrin over-expressed tumor cells, and Tat48-60 modification on MSNs could

19

enhance intracellular drug delivery, exhibiting an obvious toxicity to tumor cells. The

20

multifunctional nanosystem constructed here can realize the controlled drug release,

21

and serve as a platform for designing multifunctional nanocarriers using diversified

22

bioactive peptide-based amphiphile.

23

KEYWORDS: peptide-amphiphile, mesoporous silica nanoparticle, tumor targeting,

24

redox-sensitive, drug release

that

the

mesopores could

of be

the

resulting

sealed tightly

DOX-loaded with

MSNs

ADDA-TCPP

25 2


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1

1. INTRODUCTION

2

Cancer is one of the worldwide killers to human health, and chemotherapy has

3

attracted great attention in cancer treatment.1 However, undesired side effects often

4

arise from unspecific and inefficient delivery of antitumor drugs in conventional

5

chemotherapy, which has impeded the therapeutic efficacy to a large extent.

6

Nanocarrier-based drug delivery systems (DDSs) have shown to be popular and

7

promising as an ideal device in tumor therapy for their desirable properties, including

8

active targeting ability to tumor cells, controlled release of antitumor drugs and

9

minimal side effects toward healthy organs etc.2 Among a variety of DDSs, great

10

effort has been taken for the development of porous inorganic nanoparticles, such as

11

mesoporous

12

nanographene7,8 and copper sulfide nanoparticles.9-10 Biocompatible MSNs with

13

unique properties, including large specific surface areas, high pore volumes, highly

14

ordered channels, easy surface modification and low toxicity, have been widely

15

developed into a type of excellent candidates for controlled drug delivery. Currently, a

16

variety of multifunctional MSNs have been intensively fabricated by incorporating

17

diverse kinds of nanovalves, such as peptides, nanoparticles, polymers, and

18

biomolecules, and utilized for encapsulating antitumor drugs into the mesopores

19

without premature drug release under physiological environment.3,4,11-21 However,

20

nanovalves can be removed by some specific stimuli, such as redox potential,

21

enzymatic activity, temperature, pH, light, magnetic actuation and electrostatics,

22

leading to a quick release of antitumor drugs. For example, Zhang’s group reported an

23

envelope-type DDS based on MSNs that were end-capped with multifunctional

24

peptide and β-Cyclodextrin (β-CD) via disulfide bonds, which exhibited great

25

potential for matrix metalloproteinase (MMP)-triggered tumor-specific targeting and

silica

nanoparticles

(MSNs),3,4

carbon

nanotubes

(CNTs),5,6

3



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Page 4 of 46

1

glutathione-induced controlled drug release.22 Recently, Liang and co-workers

2

reported the use of oligonucleotides containing a DNA aptamer and a double-stranded

3

DNA (dsDNA) that could self-assemble into three-dimensional nanostructures and

4

plugged the pores.23 Bräuchle and co-workers designed a reversible pH-sensitive

5

NDDS based on poly(2-vinylpyridine) (PVP)-functionalized MSNs.24 De Cola and

6

co-workers reported a kind of novel MSN endowed with disulfide linkages in its

7

framework (ss-NPs) and RGD targeting ligands, exhibiting the active tumor-targeting

8

ability, redox-sensitive self-destructive behavior and controlled drug release in the

9

presence of intracellular GSH.25

10

Among various kinds of nanovalves, peptides always have been widely utilized

11

to cap the pores of MSNs due to their distinct physical properties, such as inherent

12

good biocompatibility, biodegradability and tunable functionality, and proven to play

13

a vital role in developing efficient and safe DDSs.26-28 In particular, many peptides

14

with short amino acid sequences can be sorted into different families, such as

15

cell-penetrating peptides (CPPs), tumor-specific targeting peptides, stimuli-sensitive

16

peptides and therapeutic peptides, on account of their different functionalities and

17

have attracted great attention in recent years. For example, RGD and RGDS peptides

18

are appropriate for targeting nanoparticles to specific tumor cells.29 Tat48-60, one of the

19

protein-derived CPPs, has been reported to possess good cell penetrating and

20

endosomal

21

arginine/lysine-rich peptide in the surface of the nanoparticles could promote

22

internalization into any cells via electrostatic interactions with the negatively charged

23

cellular membrane, even in the absence of overexpressed integrin receptors.

24

Moreover, the conjugation of hydrophobic moieties to Proline-rich peptides has been

25

reported to perform interesting self-assembly properties and more superior

escape

abilities,

since

the

presence

of

positive

charges

of

30,31

4


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1

cellular-membrane translocation abilities.31 Thus, the designed peptide-amphiphile

2

that comprises of hydrophobic alkyl chain ADDA and amphipathic Tat48-60 peptide

3

rich in Proline tends to form the self-assembly more easily. As traditional antitumor

4

drugs cannot distinguish between normal cells and tumor cells, multifunctional

5

peptides that combine properties of tumor-specific targeting and cellular membrane

6

penetrating have been designed as smart nanovalves on MSNs, endowing MSNs with

7

ability of specific binding to the specific receptors, which is overexpressed on tumor

8

cells, and thus entering the tumor cells effectively.11,12,14,22,32

9

In this work, we report on a simple, but effective self-assembly strategy to

10

fabricate a redox-sensitive DDS based on RGDS sequence containing peptide-

11

amphiphile-plugged MSNs, named DOX@MSN-ss-ADDA-TCPP. The surface

12

conjugated ADDA-TCPP mainly consist of two segments: a hydrophobic alkyl chain

13

ADDA and a hydrophilic amino acid sequence containing a Tat48-60 peptide sequence

14

with a thiol terminal group and an RGDS targeting ligand, which could spontaneously

15

self-assemble into the nanovalve of DOX@MSN-ss-ADDA-TCPP, as depicted in

16

Scheme 1A. Moreover, the conjugation of ADDA-TCPP would allow the pores of

17

MSNs to be sealed firmly, and facilitate the tumor cellular membrane internalization.

18

As

19

(DOX@MSN-ss-ADDA-TCPP) were incubated with normal cells and tumor cells,

20

they would accumulate in specific tumor cells via αvβ3-integrin receptor-mediated

21

endocytosis, followed by efficiently penetrating cellular membrane and entering the

22

cell. However, the nanovalves would be removed after the breakage of disulfide

23

bonds under the stimulus of GSH in the cellular cytoplasm, leaving pores of

24

DOX@MSN-ss-ADDA-TCPP to an open state and thus inducing an accelerated drug

25

release. The controllable drug release behavior and targeted drug delivery of

illustrated

in

Scheme

1B,

once

redox-sensitive

DOX-loaded

MSNs

5



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1

DOX@MSN-ss-ADDA-TCPP in tumor cells were further investigated, proving that

2

our proposal here could provide an appealing platform using self-assemblies of

3

peptide-based amphiphile as responsive nanovalves.

4 5 6

2. EXPERIMENTAL SECTION 2.1.

N-Cetyltrimethylammonium

Materials.

bromide

(CTAB),

7

tetraethylorthosilicate(TEOS), piperidine, ninhydrin, dichloromethane (DCM), phenol

8

and trifluoroacetic acid (TFA) were received from Shanghai Reagent Chemical Co. and

9

used directly. N,N-Dimethylformamide (DMF) was received from Shanghai Reagent

10

Chemical Co. and used after distillation. 2-Chlorotrityl chloride resin (100~200 mesh,

11

0.537

12

trimethoxysilane

13

carbodiimide(EDC·HCl) were purchased from Sigma-Aldrich Reagent and used as

14

received.

15

(Fmoc-Gly-OH、Fmoc-Ser(tBu)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gln(Trt)-OH、

16

Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Arg(Pbf)-OH and Fmoc-Pro-OH),

17

1,2-ethanedithiol

18

o-benzotriazol-N,N,N',N'-tetramethyluronium

19

N-hydroxybenzotriazole(HOBt) and triisopropylsilane (TIS) were acquired from GL

20

Biochem Ltd and used directly. (3-mercaptopropyl) trimethoxysilane (MPTMS) and

21

2,2'-dipyridyl disulfide were obtained from Aladdin Reagent Co. Ltd. and used

22

directly. 12-aminododecanoic acid (ADDA) was received from ACROS (USA) and

23

used directly. Doxorubicin hydrochloride (DOX) was obtained from Zhejiang Hisun

24

Pharmaceutical Co. (China).

25

mmol/g),

N-hydroxysuccinimide(NHS), (AAPTMS)

and

1-ethyl-3-(3-dimethly-aminopropyl)

N-fluorenyl-9-methoxycarbonyl

(EDT),

γ-(2-aminoethyl)-aminopropyl

(FMOC)

protected

L-amino

diisopropylethylamine hexafluorophosphate

acids

(DiEA), (HBTU),

Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), 6


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1

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

2

penicillin-streptomycin, Dulbecco’s phosphate buffered saline (PBS), fluorescein

3

isothiocyanate (FITC) and fluorescent molecular probe (Hoechst 33342) were

4

purchased from Invitrogen. African green monkey SV40-transformed kidney fibroblast

5

cells (COS7) and Cervical cancer cells (HeLa) were obtained from Cell Bank of

6

Chinese Academy of Sciences (Shanghai, China). All other reagents and solvents were

7

of analytical grade and used directly.

8

2.2. Characterization. The morphology of peptide-amphiphile ADDA-TCPP,

9

blank MSNs and drug-loaded MSNs were observed under a JEOL-2100F

10

transmission electron microscope (TEM) operating at 200 kV. The hydrodynamic size,

11

size distribution and zeta potential of different nanoparticles were measured by

12

dynamic light scattering (DLS) using a Zetasizer Nano ZSP (Nano-ZSZEN 3600) at

13

37 °C, and measurements for each sample were conducted with 30 sub-runs in

14

triplicate. The specific surface area of different nanoparticles was determined by

15

Brunauere Emmette Teller (BET) measurements and their corresponding pore size

16

distribution

17

Micromeritics) analysis, and all samples were pretreated at 120oC for 6 h under

18

nitrogen atmosphere before tests. FTIR spectra were carried out on a Perkin-Elmer

19

infrared spectrophotometer over the range from 4000 to 500 cm−1 employing

20

potassium bromide (KBr) technique. The structural properties of blank MSNs were

21

characterized by Low angle X-ray powder diffraction (XRD) analysis on an X’ Pert

22

Prodiffractometer (PAN alytical). Thermal gravitational analysis (TGA) data were

23

recorded on a Perkin Elmer Thermo Gravimetric Analyzer from 50 to 800 °C in a

24

nitrogen flow at a heating rate of 10 °C/min. X-Ray photoelectric spectroscopy (XPS)

25

was operated on a ESCALAB 250Xi spectrometer (Thermo Fisher) with Al Kαsource

was

detected by

Barrete-Joyner-Halenda

(BJH) (ASAP 2020,

7



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1

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(1486.6 eV) at 14.0 KV.

2

2.3. Solid Phase Synthesis of Peptides. All peptides were synthesized manually

3

on the 2-chlorotrityl chloride resin (100~200 mesh, 0.537 mmol/g) through

4

Fmoc-based solid phase peptide synthesis (SPPS) method as our previous work has

5

reported.11 The resin was washed with DCM and DMF three times respectively, and

6

soaked in DMF for 30 min. Subsequently, ADDA was coupled to the resin using 4

7

equivalent (relative to the resin loading) of ADDA and 6 equivalent of DiEA, and

8

reacting for 2 h at room temperature. After draining off the reaction solution and

9

washing the resin with DMF several times, following peptide couplings were proceed

10

with 4 equivalent of Fmoc protected amino acid, HOBT, HBTU and 6 equivalent of

11

DiEA for 3 h. Subsequently, Fmoc protected groups were cleaved by dissolving the

12

resin in a 25% piperidine/DMF (V/V) solution, and the reaction mixture was stirred

13

for 20 min at room temperature. During the synthetic process, every reaction was

14

terminated by draining off the reaction solution and washing the resin with DMF

15

several times. Furthermore, completion of the coupling reaction was proved by a blue

16

color in the Kaiser test. After the repeated acylation reaction and deprotection of

17

Fmoc groups, the target peptide sequence was obtained and fell down from the resin

18

by stirring resin with a mixture of TFA, H2O and TIS in the volume ratio of 95: 2.5:

19

2.5 for 2 h at room temperature. The cleavage mixture was concentrated and

20

precipitated in cold ether, washed with cold ether several times and dried under

21

vacuum to obtain the crude product. Then, the product was dissolved in distilled water,

22

and the solution was shifted to a dialysis bag (MWCO 1000 Da). After dialyzing

23

against distilled water for 3 days, the final product was obtained by lyophilization.

24

The molecular mass of SDGRQPPRRRQRRKKRGC-C11-COOH was determined by

25

matrix-assisted laser desorption/ionization time of flight mass spectrometry 8


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1

(MALDI-TOF-MS) system, and the molecular mass of RGDS was determined by

2

electrospray ionization-mass spectrometry (ESI-MS) system. MALDI-TOF-MS of

3

SDGRQPPRRRQRRKKRGC-C11-COOH, 2434.5 [M+H]+ (Supporting Information,

4

Figure S1). ESI-MS of free RGDS, 434.2 [M+H]+ (Supporting Information, Figure

5

S2).

6

2.4. Synthesis of Mesoporous Silica Nanoparticles (MSNs). In order to prepare

7

MCM-41 type MSNs, 1.0 g of CTAB and 0.28 g of NaOH were first dissolved in 480

8

mL of deionized water, and the solution was heated to 80 oC under vigorously stirring

9

for 15 min. After then, 5.0 g of TEOS was added to the mixture dropwise and stirred

10

vigorously for another 2 h, completing the formation of MSNs. The product was

11

obtained by centrifugation (8500 rpm/min), washing with deionized water (four times)

12

and methanol (four times), and further vacuum drying. The residual CTAB was

13

removed by refluxing MSNs in a mixture of methanol (32 mL) and HCl (2 mL) at 60

14

o

15

min), washing with deionized water (four times) and methanol (four times), and finally

16

vacuum drying.

C for 48 h. The final product was obtained by centrifugation (10000 rpm/min, 10

17

2.5. Synthesis of MSN-SH. 500 mg of blank MSNs was dispersed in a mixture

18

of MPTMS (3 mL) and methanol (40 mL), and stirred overnight at room temperature.

19

Afterwards, the MSN-SH were collected by centrifugation (10000 rpm/min, 10 min),

20

washing with deionized water (four times) and methanol (four times), and further

21

vacuum drying. The residual CTAB was removed by refluxing MSN-SH

22

nanoparticles in a mixture of methanol (32 mL) and HCl (2 mL) at 60 oC for 48 h. The

23

final product was obtained by centrifugation (10000 rpm/min, 10 min), washing with

24

methanol several times, and finally vacuum drying.

25

2.6. Synthesis of MSN-ss-Pyridyl. 500 mg of MSN-SH with CTAB template 9



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Page 10 of 46

1

extracted was dispersed in a mixture of 2,2’-dipyridyl disulfide (80 mg) and methanol

2

(20 mL), and stirred in dark for 24 h at room temperature. The final product was

3

obtained by centrifugation (10000 rpm/min, 10 min), washing with methanol several

4

times, and further vacuum drying.

5

2.7.

Drug

Loading

and

Synthesis

of

Bare

MSNs

without

the

6

Peptide-Amphiphile Cap (DOX@MSN). DOX was loaded into MSNs by

7

suspending 100 mg of MSN-ss-Pyridyl in 10 mL of PBS buffer (pH 7.4, 10 mM) at a

8

DOX concentration of 1 mg/mL, and left to stir in dark at room temperature for 24 h,

9

ensuring that DOX was entrapped into the mesopores to a great extent. After then, the

10

resulting DOX@MSN were collected by centrifugation (10000 rpm/min, 10 min),

11

washing with PBS buffer (pH 7.4, 10 mM) several times, and finally vacuum drying.

12

2.8. Drug Loading and Synthesis of Peptide-Amphiphile Gated MSNs via

13

Disulfide Bonds (DOX@MSN-ss-ADDA-TCPP). DOX was loaded into MSNs by

14

suspending 100 mg of MSN-ss-Pyridyl in 10 mL of PBS buffer (pH 7.4, 10 mM) at a

15

DOX concentration of 1 mg/mL, and left to stir in dark at room temperature for 24 h,

16

ensuring that DOX was entrapped into the mesopores to a great extent. After then,

17

100mg of ADDA-TCPP was added and the mixture was stirred in dark at room

18

temperature for 24 h to facilitate successful drug loading and capping of the

19

peptide-amphiphile nanovalve. The resulting DOX@MSN-ss-ADDA-TCPP were

20

collected by centrifugation (10000 rpm/min, 10 min), washing with PBS buffer (pH

21

7.4, 10 mM) several times, and finally vacuum drying.

22

2.9. Synthesis of MSN-NH2 and FITC-MSN-NH2. 500 mg of Blank MSNs was

23

dispersed in a mixture of AAPTMS (3 mL) and methanol (40 mL), and stirred

24

overnight at room temperature. Afterwards, the MSN-NH2 was collected by

25

centrifugation (10000 rpm/min, 10 min), washing with deionized water (four times) 10


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1

and methanol (four times). The residual CTAB was removed by refluxing the mixture

2

of methanol (32 mL) and HCl (2 mL) containing MSN-NH2 at 60 oC for 48 h. The

3

final product was obtained by centrifugation (10000 rpm/min, 10 min), washing with

4

methanol several times, and further vacuum drying.

5

For

the

preparation

of

FITC

labeled

MSN-NH2 (FITC-MSN-NH2),

6

FITC-APTMS was firstly synthesized via the reaction between AAPTMS (1 mL) and

7

FITC (5 mg) in methanol (10 mL) for 6 h at room temperature in the dark. After then,

8

Blank MSNs (100 mg) were dispersed in a mixture of FITC–AAPTMS (1 mL) and

9

methanol (10 mL), and stirred overnight at room temperature, and the other

10

procedures were conducted as same as for the synthesis of MSN-NH2.33

11

2.10. Synthesis of Fluorescein Labeled MSNs (FITC-MSN-ADDA-TCPP).

12

100 mg of FITC-MSN-NH2 nanoparticles was dispersed in 10 mL of PBS buffer (pH

13

7.4, 10 mM) and stirred at room temperature. Subsequently, 100 mg of ADDA-TCPP,

14

38.3 mg of EDC and 57.5 mg of NHS were dissolved in 5 mL of PBS buffer (pH 7.4,

15

10 mM), and stirred at 4 oC for 30 min to activate the carboxylic group of

16

ADDA-TCPP. After then, the above solution was added to 10 mL of PBS buffer

17

containing FITC-MSN-NH2 and stirred for another 24 h, ensuring the successful

18

nanovalve capping of FITC-MSNs via amide bonds. The resulting nanoparticles

19

(FITC-MSN-ADDA-TCPP) were collected by centrifugation (10000 rpm/min, 10

20

min), washing with PBS buffer (pH 7.4, 10 mM) several times, and further vacuum

21

drying.

22

2.11. Drug Loading and Synthesis of Peptide-Amphiphile Gated MSNs via Bonds

(DOX@MSN-ADDA-TCPP).

Nonredox-sensitive

23

Non-Disulfide

24

drug-loaded nanoparticles (DOX@MSN-ADDA-TCPP) were synthesized as control.

25

Briefly, 100 mg of MSN-NH2 nanoparticles was dispersed in 10 mL of PBS buffer 11



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Page 12 of 46

1

(pH 7.4, 10 mM) at a DOX concentration of 1mg/mL and left to stir in dark at room

2

temperature for 24 h, ensuring that DOX was entrapped into the mesopores to a great

3

extent. Subsequently, 100 mg of ADDA-TCPP, 38.3 mg of EDC and 57.5 mg of NHS

4

were dissolved in 5 mL of PBS buffer (pH 7.4,10 mM), and stirred at 4 oC for 30 min

5

to activate the carboxylic group of ADDA-TCPP. After then, the above solution was

6

added to 10 mL of PBS buffer containing drug-loaded DOX@MSN-NH2 and stirred

7

for another 24 h, ensuring the successful nanovalve capping of drug-loaded MSNs via

8

amide bonds. The resulting nanoparticles (DOX@MSN-ADDA-TCPP) were

9

collected by centrifugation (10000 rpm/min, 10 min), washing with PBS buffer (pH

10 11

7.4,10 mM) several times, and further vacuum drying. 2.12.

Investigation

of

Molecular

Self-Assembly

Behaviour

of

12

Surface-Conjugated ADDA-TCPP. 20 mg of ADDA-TCPP peptide-amphiphile

13

grafted MSNs was treated with 10% HF solution (v/v), and stirred for 3 hours at room

14

temperature, ensuring the complete etching of the inner silica core from the peptidic

15

shell. Subsequently, the degrafted ADDA-TCPP chains from MSN samples were

16

dialyzed against distilled water and obtained by lyophilization before the critical

17

micelle concentration (CMC) measurement.

18

The CMC analysis of peptide-amphiphile ADDA-TCPP was performed by using

19

pyrene as a hydrophobic fluorescent probe. Briefly, 0.1 mL of acetone solution

20

containing pyrene (6×10-6 M) was added to tubes, and acetone was acquired to be

21

evaporated completely.34 Subsequently, 1 mL of aqueous solution containing various

22

amounts of ADDA-TCPP was added to the tubes, and shaken gently at 37 °C

23

overnight. After then, the solution above was transferred to aquartz cell, and

24

excitation spectra data were recorded over the range from 300 to 360 nm, with

25

emission wavelength of 390 nm. On the basis of the excitation spectra of pyrene, the 12


Page 13 of 46

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1

fluorescence intensity ratios (I337/I334) were plotted against the logarithm of the

2

ADDA-TCPP concentrations. The corresponding CMC value was calculated from the

3

point of intersection, where the tangent to the curve meets with the horizontal tangent

4

at a low concentration.

5

2.13. Determination of Drug Loading Efficiency (DLE) and Drug Loading

6

Content (DLC) of Drug Loaded MSNs. Since HF solution is corrosive and can

7

induce the mesoporous silica structural collapse, 0.1 mg of DOX-loaded MSNs was

8

firstly dissolved in HF solution (0.1 M), and NaOH solution (0.1M) was added to

9

adjust pH value of the dissolution to ~7.4 for direct fluorescence detection.35 DOX

10

fluorescence intensity of the dissolution was obtained by using a RF-530/PC

11

spectrofluorophotometer (Shimadzu) with an excitation at 480 nm, and DOX

12

concentration was calculated by a fluorescence standard calibration curve. The DLE was defined as follow: DLE = (weight of drug loaded in MSNs/weight

13 14

of drug loaded MSNs) × 100%. The DLC was defined as follow: DLC= (weight of drug loaded in MSNs/weight

15 16

of feed drug) × 100%. 2.14. Evaluation of DOX Release Behaviors in Vitro. Drug release studies

17 18

were

carried

out

on

different

kinds

of

DOX-loaded

MSNs

samples

19

(DOX@MSN-ss-ADDA-TCPP, DOX@MSN-ADDA-TCPP, and DOX@MSN) in

20

PBS solution (pH 7.4, 10 mM) containing different concentration of GSH (0 mM, 2

21

µM and 10 mM), demonstrating the redox-sensitive ability of the corresponding

22

nanovalve. In brief, 5 mg of drug loaded nanoparticles was evenly suspended in 2 mL

23

of PBS solution (pH 7.4, 10 mM) at different GSH concentration (0 mM, 2 µM and

24

10 mM). After then, the suspension (2.5 mg/mL) was transferred to a dialysis bag

25

(MWCO 12000 Da), immersed into 10 mL of PBS solution at different GSH 13



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Page 14 of 46

1

concentration (0 mM, 2 µM and 10 mM), and shaken slowly at 37 oC. At specific time

2

intervals, 3 mL of external PBS solution was replenished with an equivalent volume

3

of fresh medium. Moreover, drug release behaviour of DOX@MSN-ss-ADDA-TCPP

4

in simulated physiological environment was operated according to the previous

5

report.36 Specifically, 5 mg of drug loaded nanoparticles was evenly suspended in 2

6

mL of distilled water prior to mixing with serum solution at pH 7.4. Then,

7

DOX@MSN with a final concentration of 1.25 mg/mL were dispersed in FBS (50%,

8

v/v), and immersed into 10 mL of FBS. At specific time intervals, 3 mL of external

9

serum solution was replenished with an equivalent volume of fresh FBS. The released

10

amount of DOX from drug loaded nanoparticles was determined by operating a

11

RF-5301PC spectrofluorophotometer (Shimadzu) with emission wavelength at 555

12

nm under the excitation was wavelength of 480 nm and calculating via a standard

13

calibration curve experimentally obtained.

14

The

cumulative

DOX

release

was

calculated

as

follow:

n -1

15

Cumulative Release(%) = (Ve ∑ i C i +V0 C n )/m × 100

16

Where Ve is the volume of external PBS solution extracted at specific time point; V0

17

is the total volume of external PBS solution; Ci is the DOX concentration of external

18

PBS solution at specific time point; m is the amount of DOX loaded in drug-loaded

19

nanoparticles and n is the specific time point.

20

2.15. Cell Culture. Tumor cells (HeLa cells) and normal cells (COS7 cells) were

21

cultured in DMEM medium with 1% penicillinestreptomycin (10,000 U/mL) and 10%

22

FBS, and incubated at a humidified atmosphere containing 5% CO2.

23

2.16. Cellular Uptake Experiment. Confocal Laser Scanning Microscopy

24

(CLSM) was utilized to trace intracellular endocytosis of fluorescein labeled

25

FITC-MSN-ADDA-TCPP and redox-sensitive drug release behavior of DOX-loaded 14


Page 15 of 46

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1

MSNs (DOX@MSN-ss-ADDA-TCPP and DOX@MSN-ADDA-TCPP) within HeLa

2

cells and COS7 cells. Firstly, cells were seeded indiscs containing 1 mL of DMEM

3

medium with 1% penicillinestreptomycin (10,000 U/mL) and 10% FBS, and cultured

4

for 24 h. After then, an equal volume of DMEM containing FITC-MSN-ADDA-TCPP

5

(30

6

DOX@MSN-ADDA-TCPP (DOX: 5 µg/mL) was added to DMEM and incubated

7

with cells for further 4 h. Subsequently, the culture medium was removed and cells

8

were washed with PBS several times, and the nuclei were stained with Hoechst 33342

9

for 15 min at 37 oC. After then, the culture medium was removed and cells were

10

washed several times with PBS solution. Finally, cells were incubated in 1 mL of

11

DMEM without FBS and viewed under a CLSM.

µg/mL),

DOX@MSN-ss-ADDA-TCPP

(DOX:

5

µg/mL),

and

12

For the integrin receptor inhibition and competition research, HeLa cells were

13

pre-treated with free RGDS peptide for 30 min, and certain amount of

14

FITC-MSN-ADDA-TCPP (30 µg/mL) or DOX@MSN-ss-ADDA-TCPP (DOX: 5

15

µg/mL) was added and incubated with HeLa cells for further 4 h. After co-incubation,

16

the following treatment was handled as described above.

17

2.17. In Vitro Flow Cytometry. HeLa cells and COS7 cells were cultured in

18

6-well plates at a density of 5×104 cells/well, and incubated in 1 mL of DMEM

19

containing 1% penicillinestreptomycin (10,000 U/mL) and 10% FBS for 24 h,

20

respectively.

21

DOX@MSN-ss-ADDA-TCPP (DOX: 5 µg/mL) or DOX@MSN-ADDA-TCPP

22

(DOX: 5 µg/mL) was added and incubated with cells for further 4 h. After washed

23

with PBS several times, cells were digested by trypsin, counted and collected by flow

24

cytometry (BD FACS AriaTM III, USA). HeLa cells and COS7 cells without any

25

treatment were served as the blank control.

Subsequently,

1

mL

of

DMEM

containing

15



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Page 16 of 46

1

For the integrin receptor inhibition and competition research, HeLa cells were

2

pre-treated with free RGDS peptide (2 µM) for 30 min, and certain amount of

3

DOX@MSN-ss-ADDA-TCPP (DOX: 5 µg/mL) was added and incubated with HeLa

4

cells for further 4 h. The following treatment was carried out as described above. 2.18. In Vitro Cytotoxicity Assay. The MTT assay was applied to evaluate the

5 6

cytotoxicity

of


(DOX:

5

µg/mL)

or

7

DOX@MSN-ADDA-TCPP (DOX: 5 µg/mL) against HeLa and COS7 cells. The cells

8

were cultured in the 96-well plate (8×103 cells/well), and incubated in 100 µL of

9

DMEM containing 1% penicillinestreptomycin (10,000 U/mL) and 10% FBS for 24 h.

10

Then, DOX-loaded MSNs dispersed in 100 µL of DMEM containing 1%

11

penicillinestreptomycin (10,000 U/mL) and 10% FBS was added to the 96-well plate

12

at different concentrations, and incubated with cells for further 48 h. Subsequently, the

13

culture medium was replaced with 200 µL of fresh DMEM containing 10% FBS,

14

followed by adding 20 µL of MTT solution. After 4 h incubation time, the culture

15

medium was removed, and 150 µL of DMSO was added to each well. The optical

16

density (OD) at 570 nm was measured by the microplate reader (Model 550,

17

BIO-RAD, USA). Finally, the mean value of eight independent experiments was

18

obtained and the relative cell viability was calculated as follows: Viability (%)=(ODtreated / ODcontrol) × 100

19 20

where the ODcontrol is acquired in the absence of DOX-loaded nanoparticles and

21

ODtreated is acquired in the presence of DOX-loaded nanoparticles. For the integrin receptor inhibition research, HeLa cells were pre-treated with

22 23

free

RGDS

peptide

(2

µM)

for

30

min,

and

certain

amount

of

24

DOX@MSN-ss-ADDA-TCPP (DOX: 5 µg/mL) was added and incubated with HeLa

25

cells. After co-incubation for further 4 h, the following treatment was carried out as 16


Page 17 of 46

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1

described above.

2

2.19. Statistics Analysis. The statistical analysis was carried out by three-sample

3

Student's text unless otherwise noted, and the quantitative data obtained were shown

4

as mean ± standard deviation (S. D). Statistical significance was considered at a value

5

of P < 0.05 and were indicated where appropriate.

6 7

3. RESULTS AND DISCUSSION

8

3.1. Fabrication and Characterization. Detailed synthetic process of the

9

redox-sensitive MSN-ss-ADDA-TCPP nanosystem was presented in Scheme S1. The

10

MCM-41 type MSNs were first synthesized according to the literature with CTAB as

11

surfactant templating agent and TEOS as silica source.21 After then, the outer surface

12

of MSNs was modified with thiol groups after their reaction with MPTMS, followed

13

by extracting CTAB to form empty mesoporous channels, which was necessary to

14

ensure effective drug loading of MSNs. The template-free MSN-SH were

15

subsequently react with 2, 2'-dipyridyl disulfide through disulfide linkages to obtain

16

an active intermediate MSN-ss-Pyridyl, according to a previously reported method.11

17

Subsequently, DOX was loaded in the pores of MSN-ss-Pyridyl by free diffusion, and

18

a peptide-amphiphile containing cysteine, ADDA-TCPP, was attached to MSN via

19

disulfide exchange reaction between ADDA-TCPP and MSN-ss-Pyridyl, constructing

20

a novel redox-sensitive nanovalve gated MSN for tumor therapy. Under neutral

21

condition, the surface-conjugated ADDA-TCPP could form molecular self-assemblies

22

through hydrophobic forces between the alkyl chains of ADDA-TCPP, and thus acted

23

as redox-sensitive nanovalves for sealing the pores of DOX@MSN-ss-ADDA-TCPP

24

tightly, as depicted in Scheme 1A.

25

According to TEM measurements (Figure 1A and B), the monodispersed blank 17



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Page 18 of 46

1

MSNs displayed a uniform and spherical morphology with hexagonally arranged pore

2

structure, and the average particle size of unmodified pristine MSNs was around 118

3

nm. Besides, three well-defined diffraction peaks in the XRD pattern further

4

confirmed the well-ordered hexagonal mesostructure of blank MSNs (Figure 1D).37

5

DLS measurement revealed that the average hydrodynamic diameter of as-obtained

6

MSNs was 253.6 nm, and the polydispersity index (PDI) was 0.18 (Figure S3). XPS

7

analysis was employed to investigate MSNs before and after functionalization of thiol

8

groups. As exhibited in Figure 1E, compared with blank MSNs, the presence of S

9

besides C, Si and O in MSN-SH verified the successful modification of thiol groups.38

10

Furthermore, the amount of thiol groups conjugated on the surface of MSN-SH was

11

determined by calculating the photoemission peak area of S 2s and S 2p orbitals in the

12

XPS spectra, and the sulphur content was found to be 4.91%. It seemed that the

13

ADDA-TCPP conjugation and drug loading didn't affect the particle size of MSNs

14

(Figure 1C). The micellization behaviour of peptide-amphiphile ADDA-TCPP was

15

examined by using CMC analysis and TEM measurement. The corresponding value

16

of CMC was calculated as 50.4 mg/L (Figure 1F), confirming that the incorporation of

17

ADDA as the hydrophobic tail could strengthen the hydrophobic aggregation ability

18

of ADDA-TCPP. Moreover, a uniform and well-dispersed spherical morphology of

19

ADDA-TCPP was observed in TEM images (Figure 1G), further demonstrating the

20

self-assembly behaviour of ADDA-TCPP superior to serve as “nanovalve” of MSNs.

21

The whole synthetic procedure was further monitored with several methods, including

22

zeta potential analysis (Table 1), nitrogen adsorption measurement (Figure 2), FT-IR

23

spectroscopy (Figure 3) and TGA (Figure 4). As shown in table 1, zeta potential of

24

MSNs, MSN-SH, MSN-ss-Pyridyl and DOX@MSN-ss-ADDA-TCPP was −25.9mV,

25

−15.7mV, -11.4 mV and +15.5 mV, respectively, and the increased zeta potential of 18


Page 19 of 46

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1

DOX@MSN-ss-ADDA-TCPP indicated that the positively charged ADDA-TCPP was

2

successfully introduced onto the surface of MSNs.

3

BET and BJH measurements were performed to investigate surface

4

functionalization of MSNs. As revealed in Figure 2A, a typical type-IV isotherm

5

curve indicated that MSNs possessed well-defined mesoporous structure. It could be

6

seen from Table S1 that an obvious decline in BET surface area (SBET), pore volume

7

(V ), and BJH pore diameter (V ) of MSNs occurred during the stepwise P

BJH

8

functionalization process. The specific surface area, pore volume and pore diameter of

9

bare MSN was 1130.69 m2/g, 1.26 cm3/g and 3.9 nm respectively, indicating that

10

these nanoparticles were ideal nanocarries for surface multi-functionalization and

11

hosting more guest molecules of diverse size. However, the specific surface area of

12

DOX@MSN-ss-ADDA-TCPP shrunk to 7.96 m2/g, confirming the successful drug

13

loading and completely blocking of pores by ADDA-TCPP self-assemblies (Figure

14

2B).

15

From FTIR spectra (Figure 3), compared with bare MSNs, MSN-SH displayed a

16

characteristic peak of thiol group at 2560 cm-1, and two absorbance bands at 2855 and

17

2926 cm−1 corresponding to C-H stretching vibrations of the alkyl chain of CTAB

18

disappeared after CTAB extraction.22,39 Moreover, successful modification of

19

ADDA-TCPP on the MSN-ss-Pyridyl was confirmed by disappearance of the typical

20

signal at 2560 cm-1 in the FT-IR spectra.

21

TGA was employed to confirm successful surface modification of MSNs and

22

determine the amount of surface coverage. As Figure 4A shown, the weight loss of

23

blank MSNs, MSN-SH, MSN-NH2, MSN-ss-Pyridyl, DOX@MSN-ss-ADDA-TCPP

24

and DOX@MSN-ADDA-TCPP was 9.28%、16.63%、18.66%、20.09%、27.45% and

25

23.04%, when heating to 800 °Cat a N2 atmosphere. As expected, the higher weight 19



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Page 20 of 46

1

loss after each modification further confirmed the successful fabrication of the

2

functionalized

3

DOX@MSN-ADDA-TCPP and DOX@MSN nanoparticles was calculated to be 4.2%,

4

4.0% and 2.1% respectively by fluorescence analysis.

MSNs.

Moreover,

DLC

of

DOX@MSN-ss-ADDA-TCPP,

5

Furthermore, detailed synthesis procedure of DOX@MSN-ADDA-TCPP

6

without disulfide linkages was depicted in Scheme S-2. MCM-41 type MSNs were

7

prepared and modified with amine groups by reacting with AAPTMS, and the

8

surfactant templating agent CTAB was extracted to obtain MSN-NH2. The

9

conjugation of ADDA-TCPP on the outer surface of MSNs was achieved through

10

amide reaction, and the successful synthesis was confirmed by TGA measurement

11

(Figure 6B). Moreover, an obvious decrease in the surface area and N2 volume

12

adsorbed was a good indicative of mesoporous systems with well-filled mesopores

13

(Figure

14

DOX@MSN-ADDA-TCPP.

S4),

further

confirming

the

successful

construction

of

15

3.2. In Vitro GSH-induced Drug Release Research. As well-known, the

16

concentration of GSH (1-2 µM) is minimal in the blood, but nearly 103 times higher

17

than that of GSH (2~10 mM) in the cellular cytoplasm.38 Moreover, the concentration

18

of GSH in some tumor tissues has been discovered to be several times higher than that

19

in normal tissues.39,40 In order to investigate redox-sensitive property of

20

DOX@MSN-ss-ADDA-TCPP with peptide-amphiphile nanovalves through disulfide

21

linkages, drug release behaviour of DOX@MSN-ss-ADDA-TCPP in PBS solution

22

(pH 7.4, 10 mM) with different concentration of GSH (0 mM, 2 µM and 10 mM) was

23

performed, and drug release behaviour of DOX@MSN-ADDA-TCPP and

24

DOX@MSN

25

DOX@MSN-ss-ADDA-TCPP were performed as control. Figure 7A showed that

under

different

conditions

as

same

as

that

of

20


Page 21 of 46

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1


2

premature drug release” prior to high concentration of GSH, demonstrating the well

3

pore-blocking ability of ADDA-TCPP nanovalves in sealing off the mesopores. In

4

addition, DOX@MSN-ss-ADDA-TCPP (Figure S5) exhibited “zero premature drug

5

release” in simulated blood solution for that the blood is much more complicated than

6

PBS buffer, further demonstrating the excellent pore-blocking ability of ADDA-TCPP

7

nanovalves.

8

DOX@MSN-ss-ADDA-TCPP was observed under exposure to 10 mM GSH. This

9

phenomenon could be attributed to the reducible cleavage of disulfide bonds triggered

10

by the high concentration of GSH. While DOX@MSN-ADDA-TCPP without

11

disulfide linkages exhibited negligible drug release, when exposed to PBS solution

12

with different concentration of GSH (Figure 7B). Moreover, DOX@MSN (Figure S6)

13

with no cap exerted cumulative drug release under different conditions (the

14

concentration of GSH in PBS was 0 mM, 2 µM and 10 mM), which was attributed to

15

the electrostatic interaction between the negative charged surface of MSN and the

16

positive charged DOX,43 as well as the drug release kinetics on pore size which have

17

been reported in the previous literatures.44,45 These results indicated that

18

DOX@MSN-ss-ADDA-TCPP containing disulfide linkages was highly sensitive to

19

intracellular GSH concentration, but keep ‘‘stealth’’ in extracellular environment

20

without DOX leakage.

However,

a

containing

dramatically

disulfide

linkages

accelerated

drug

exhibited

release

“zero

from

21 22

3.3.

Tumor

Targeting

Ability

Mesoporous

of Silica

Multifunctional

23

Peptide-Amphiphile-Capped

Nanoparticles

24

(MSN-ADDA-TCPP). To demonstrate the targeting effect of the RGDS motif upon

25

intracellular endocytosis of MSN-ADDA-TCPP, the green fluorescent dye FITC was 21



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Page 22 of 46

1

tagged on the surface of MSN-ADDA-TCPP to obtain fluorescein labeled

2

FITC-MSN-ADDA-TCPP. As shown in Figure 6A1-A4, a weak green fluorescence

3

was observed in COS7 cells after 4 h incubation with FITC-MSN-ADDA-TCPP.

4

However, a strong green fluorescence was observed in HeLa cells after 4 h incubation

5

with

6

functionalized MSNs were taken up by the tumor cells efficiently. This was attributed

7

to the exposed RGDS motif on FITC-MSN-ADDA-TCPP with an excellent ability to

8

selectively bind to overexpressed receptors, and resulted in an efficient internalization

9

of nanoparticles towards tumor cells. To further assess the significant role played by

10

the conjugated RGDS peptide in the tumor cellular internalization, HeLa cells were

11

pre-incubated with free RGDS peptide as competitors for 30 min, followed by being

12

treated with FITC-MSN-ADDA-TCPP for 4 h, and then observed under CLSM. As

13

presented in Figure 6C1-C4, an evidently weakened FITC green fluorescence in HeLa

14

cells clearly revealed that free RGDS could compete with surface-conjugated RGDS

15

of drug-loaded MSNs in binding with the surface integrin receptor-overexpressed

16

HeLa cells, and therefore significantly inhibited the cellular uptake.

FITC-MSN-ADDA-TCPP

(Figure

6B1-B4),

demonstrating

that

the

17

3.4. Intracellular Drug Delivery of DOX@MSNs-ss-ADDA-TCPP Observed

18

by CLSM. To evidence the critical role displayed by the surface-conjugated

19

ADDA-TCPP nanovalve of drug-loaded MSNs during the endocytosis process,

20

CLSM was employed to observe the cellular uptake and subsequent drug delivery of

21

DOX@MSNs-ss-ADDA-TCPP, and the cellular uptake and subsequent drug delivery

22

of DOX@MSNs-ADDA-TCPP were performed as control. As CLSM images shown

23

(Figure 7B1-B4), a strong DOX red fluorescence distributed in cytoplasm of HeLa

24

cells, indicating that RGDS peptide could mediate the most efficient delivery of

25

DOX@MSNs-ss-ADDA-TCPP to HeLa cells, followed by enhanced drug release 22


Page 23 of 46

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1

triggered by intracellular GSH. However, we found that the red fluorescence intensity

2

of DOX got very weak in both COS7 cells (Figure 7A1-A4) and HeLa cells incubated

3

in presence of free RGDS (Figure 7C1-C4). These results clearly revealed that normal

4

cells that express αvβ3 integrin in low level showed an extremely limited

5

internalization, due to the lack of specific cell recognition. Moreover, free RGDS

6

could compete with surface-conjugated RGDS of drug-loaded MSNs in binding with

7

the surface integrin receptor-overexpressed HeLa cells, and therefore significantly

8

inhibited the cellular uptake. What’s more, little DOX fluorescence was observed

9

from CLSM images when incubated DOX@MSNs-ADDA-TCPP with both COS7

10

cells (Figure 8A1-A4) and HeLa cells in the absence (Figure 8B1-B4)/presence (Figure

11

8C1-C4) of free RGDS peptide, which was due to the nonredox-sensitive nanovalve

12

capping of DOX@MSNs-ADDA-TCPP exhibiting nearly zero drug release inside the

13

cell.

14

Furthermore, flow cytometry analysis was employed to quantitatively determine

15

intracellular uptake of DOX-loaded MSNs in the presence or absence of free RGDS.

16

As shown in Figure 7D-E, the mean fluorescence intensity (MFI) of HeLa cells

17

(21700) incubated with the redox-sensitive DOX@MSNs-ss-ADDA-TCPP almost

18

reached 3 times higher than that of COS7 cells (7272). However, the MFI of HeLa

19

cells (8149) dramatically decreased upon exposure to free RGDS peptide. Besides, the

20

nonredox-sensitive DOX@MSNs-ADDA-TCPP did not induce a significant

21

difference among the MFI of HeLa cells in the absence and presence of free RGDS

22

peptide and that of COS7 cells (Figure 8D-E). The flow cytometry analysis above was

23

in consistence with CLSM observations regarding the specific tumor targeting and

24

redox-sensitive drug delivery, further confirmed that RGDS peptide was responsible

25

for

effective

tumor

targeting

ability

of

the

redox-sensitive 23



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Page 24 of 46

1

DOX@MSNs-ss-ADDA-TCPP. These results suggested that existence of RGDS

2

motif and disulfide linkages on the surface of drug delivery nanosystem could

3

enhance the cellular uptake and facilitate drug release in response to intracellular

4

reductive environment.

5

3.5. In Vitro Cytotoxicity Assay. The in vitro cytotoxicity of blank MSNs and

6

drug-loaded MSNs against HeLa cells and COS7 cells was further confirmed by MTT

7

analysis. As shown in Figure 9A, blank MSNs displayed low cytotoxicity that less

8

than 10% of both HeLa cells and COS7 cells died even at high concentration of 100

9

µg/mL after 48 h incubation, demonstrating a good biocompatibility of blank MSNs.

10

However,

the

11

cytotoxicity against HeLa cells than COS7 cells under the same situation (Figure 9B).

12

It was mainly owned to the specific recognition of surface integrin receptor

13

over-expressed HeLa cells by surface-conjugated RGDS peptide of DOX-loaded

14

MSNs, and a large amount of DOX released from the disulfide nanovalve plugged

15

DOX@MSNs-ss-ADDA-TCPP under the stimulus of GSH in the cellular cytoplasm.

16

Comparing with the redox-sensitive DOX@MSNs-ss-ADDA-TCPP containing

17

disulfide nanovalves, the nonredox-sensitive DOX@MSNs-ADDA-TCPP showed

18

obviously reduced cytotoxicity. As presented in Figure 9C, the cell viability of

19

nonredox-sensitive DOX@MSNs-ADDA-TCPP was still over 60% even at the high

20

DOX concentration (5 µg/mL), which probably due to the non-cleavage of the

21

nonredox-sensitive nanovalve and thus minimal leakage of DOX was occured from

22

DOX@MSNs-ADDA-TCPP in the cytoplasm. The half-inhibitory concentration (IC50)

23

of

24

DOX@MSNs-ADDA-TCPP) was concluded in Table 2. The IC50 value of

25

DOX@MSN-ss-ADDA-TCPP in HeLa cells was ~0.7 µg/mL, which was nearly 3

drug

redox-sensitive

loaded


MSNs

exerted

(DOX@MSNs-ss-ADDA-TCPP

higher

and

24


Page 25 of 46

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1

times lower than that in COS7 cells (∼2.0 µg/mL). Furthermore, the introduction of

2

free RGDS evaluated the IC50 value of DOX@MSN-ss-ADDA-TCPP in HeLa cells

3

(1.7 µg/mL) to nearly 2.4 times higher than that of DOX@MSN-ss-ADDA-TCPP in

4

the absence of free RGDS, confirming the competition existed between free RGDS

5

and the RGDS sequence on the surface of drug-loaded MSNs. In contrast with

6

redox-sensitive MSNs, the IC50 value of DOX@MSN-ADDA-TCPP without

7

redox-sensitive linkages was not acquired, further verifying the perfect blockage of

8

ADDA-TCPP nanovalve. The result demonstrated that the incorporation of disulfide

9

nanovalves could control the release of DOX in cytosolic environments, implying a

10

great potential for application of the peptide-amphiphile nanovalve capped MSN as a

11

smart drug nanocarrier in an effective tumor therapy.

12 13

4. CONCLUSIONS

14

Currently, DOX is commonly applied in early clinical stages of cervical cancer

15

treatment.46,47 However, the selective targeting of cancer cells and controlled drug

16

release remain a significant challenge in successful chemotherapy. To address these

17

troubles, here, we have designed and constructed a novel drug delivery platform based

18

on multifunctional peptide-amphiphile nanovalves. In vitro drug release investigations

19

demonstrated that DOX could be sealed in the pores firmly with nearly no leakage in

20

the absence of GSH or in the presence of low GSH concentration, realizing “zero

21

premature release” of DOX in physical environment. In vitro cell experiments

22

confirmed that redox-sensitive MSNs could internalize into tumor cells via the

23

receptor-mediated endocytosis, and the surface peptide-amphiphile nanovalve

24

ADDA-TCPP of redox-sensitive DOX@MSN-ss-ADDA-TCPP would be detached 25



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Page 26 of 46

1

due to the cleavage of disulfide bonds triggered by GSH in cellular cytoplasm,

2

inducing a fast drug release inside tumor cells. To our knowledge, it is the first time to

3

construct

4

peptide-amphiphile, and achieved good results in sealing the pores of MSNs and

5

controlled

6

(DOX@MSN-ss-ADDA-TCPP) built on self-assemblies of peptide-amphiphile

7

nanovalves gated MSNs would offer an opportunity for the development of smart

8

NDDS for effective tumor therapy.

a

nanovalve

releasing

utilizing

of

the

self-assembly

antitumor

drug.

of

This

multifunctional

novel

DDS

9 10

ASSOCIATED CONTENT

11

Supporting Information

12

The Supporting Information is available free of charge on the ACS Publications

13

website

14

BET and BJH parameters of different nanoparticles; Construction of different

15

functionalized MSNs; MALDI-TOF and ESI-MS spectra of synthesized peptides;

16

XRD pattern and size distribution of blank MSNs.

17 18

AUTHOR INFORMATION

19

Corresponding Author

20

*E-mails: [email protected] (Y. J. C.), [email protected] (X. Z.)

21

Author Contributions

22

All authors have given approval to the final version of the manuscript.

23

Notes

24

The authors declare no competing financial interest. 26


Page 27 of 46

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1

ACKNOWLEDGMENTS

2

We acknowledge the financial support from the National Natural Science Foundation

3

of China (51233003, 51303137) and the Chinese Postdoctoral Science Foundation

4

(2014T70729).

5

27



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1

Page 34 of 46

Table 1. Zeta potential of different nanoparticles in PBS (pH 7.4, 10 mM) at 37 oC. Sample

Zeta Potential (mV)

MSN

-25.9

MSN-SH

-15.7

MSN-ss-Pyridyl

-11.4


15.5

2

34


Page 35 of 46

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1

Table 2. IC50 values of DOX-loaded nanoparticles against HeLa and COS7 cells. DOX@MSN-ss-ADDA-T

DOX@MSN-ADD

DOX@MSN-ss-TPP&TCPP

CPP

A-TCPP

+ Free RGDS (mg/L)

Cell

HeLa

0.7

\

1.7

COS7

2.0

\

\

2

35



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Page 36 of 46

1 2 3

Scheme 1. (A) Self-assembly procedure of peptide-amphiphile conjugated on MSNs.

4

(B) Redox-sensitive tumor targeting drug delivery: (i) Specific uptake by tumor cells

5

through RGDS-mediated interaction; (ii) Endocytosis into specific tumor cells; (iii)

6

Glutathione-triggered drug release via breakage of disulfide bonds; (iv) Drug arrive at

7

the cytoplasm by diffusion and eventually enter into the nucleus, leading to the

8

apoptosis of tumor cells.

9

36


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1 2

Figure 1. Characterization of the multifunctional MSN. (A, B and C) TEM images

3

of blank MSN (A and B) and DOX@MSN-ss-ADDA-TCPP (C). (D) XRD analysis of

4

blank MSNs. (E) XPS for MSN and MSN-SH. (F) Intensity of I337/I334 in the

5

excitation spectra as a function of logarithm of the concentration. (G) TEM image of

6

peptide-amphiphile ADDA-TCPP.

7

37



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Page 38 of 46

1

2 3

Figure 2. BET nitrogen adsorption/desorption isotherms (A) and BJH pore size

4

distribution (B) of different nanoparticles.

5

38


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1 2

Figure 3. FT-IR spectra of MSN, MSN-SH, MSN-ss-Pyridyl and

3

DOX@MSN-ss-ADDA-TCPP.

4

39



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Page 40 of 46

1 2

Figure 4. TGA curves of multifunctional DOX@MSN-ss-ADDA-TCPP (A) and

3

DOX@MSN-ADDA-TCPP (B).

4

40


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1 2

Figure 5. In vitro drug release behavior of DOX@MSN-ss-ADDA-TCPP (A) and

3

DOX@MSN-ADDA-TCPP (B) in PBS buffer (pH 7.4, 10 mM) with different GSH

4

concentrations (0 mM, 2 µM and 10 mM) at physiological temperature (37 °C). Data

5

are shown as the mean ± SD (*p < 0.05 as compared with the data in PBS, n =3).

6

41



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Page 42 of 46

1 2

Figure 6. CLSM images of CLSM images of COS7 cells (A) and HeLa cells (B)

3

respectively incubated with FITC-MSN-ADDA-TCPP (30 µg/mL) for 4 h; (C) CLSM

4

images of HeLa cells incubated with FITC-MSN-ADDA-TCPP (30 µg/mL) in the

5

presence of free RGDS (2 µM) for 4 h. (A1, B1, C1) blue fluorescence images of

6

Hoechst 33342; (A2, B2, C2) green fluorescence images of FITC; (A3, B3, C3) overlap

7

of confocal fluorescence images; (A4, B4, C4) overlap of confocal fluorescence image

8

and bright field images. (The scale bar is 20 µm).

42


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1 2

Figure 7. CLSM images of COS7 (A) and HeLa (B) cells respectively incubated with

3

the DOX@MSN-ss-ADDA-TCPP (relative DOX concentration, 5 µg/mL) for 4 h. (C)

4

CLSM images of HeLa cells incubated with DOX@MSN-ss-ADDA-TCPP (relative

5

DOX concentration, 5 µg/mL) in the presence of free RGDS (2 µM) for 4 h. (A1, B1,

6

C1) blue fluorescence images of Hoechst 33342; (A2, B2, C2) red fluorescence images

7

of DOX; (A3, B3, C3) overlap of confocal fluorescence images; (A4, B4, C4) overlap of

8

confocal fluorescence images and bright field images. (The scale bar is 20 µm). (D)

9

Flow cytometry analysis and (E) MFI of COS7 cells (black) incubated with

10

DOX@MSN-ss-ADDA-TCPP for 4 h; MFI of HeLa cells incubated with

11

DOX@MSN-ss-ADDA-TCPP in the absence (yellow) and presence (green) of free

12

RGDS (2 µM) for 4 h. respectively; MFI of COS7 (red) and HeLa (blue) cells

13

incubated with the blank. 43



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Page 44 of 46

1 2

Figure 8. CLSM images of COS7 (A) and HeLa (B) cells respectively incubated with

3

the DOX@MSN-ADDA-TCPP nanoparticles (relative DOX concentration, 5 µg/mL)

4

for 4 h. (C) CLSM images of HeLa cells in the presence of free RGDS (2 µM) for 4 h.

5

(A1, B1, C1) blue fluorescence images of Hoechst 33342; (A2, B2, C2) red fluorescence

6

images of DOX; (A3, B3, C3) overlap of confocal fluorescence images; (A4, B4, C4)

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overlap of confocal fluorescence images and bright field images. (The scale bar is 20

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µm). (D) Flow cytometry analysis and (E) MFI of COS7 cells (black) incubated with

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DOX@MSN-ADDA-TCPP nanoparticles for 4 h; MFI of HeLa cells incubated with

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DOX@MSN-ADDA-TCPP in the absence (yellow) and presence (green) of free

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RGDS (2 µM) for 4 h. respectively; MFI of COS7 (red) and HeLa (blue) cells

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incubated with the blank.

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Figure 9. The viability of COS7 and HeLa cells incubated with blank MSNs (A),

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DOX@MSN-ss-ADDA-TCPP in the absence/presence of free RGDS peptide (2 µM)

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(B), and DOX@MSN-ADDA-TCPP (C) in the absence/presence of free RGDS

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peptide (2 µM) for 48 h. Data are shown as the mean ± S.D. (*p < 0.05, n=8).

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Page 46 of 46

Table of Contents/Abstract Graphic

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Multifunctional Peptide-Amphiphile End-Capped ... - ACS Publications

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