pH-Sensitive Delivery Vehicle Based on Folic Acid-Conjugated

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

A pH-sensitive delivery vehicle based on folic acid-conjugated polydopaminemodified mesoporous silica nanoparticles for targeted cancer therapy Wei Cheng, Junpeng Nie, Lv Xu, Chaoyu Liang, Yunmei Peng, Gan Liu, Teng Wang, Lin Mei, Laiqiang Huang, and Xiaowei Zeng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 12 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

A

pH-sensitive

delivery

vehicle

based

on

folic

acid-conjugated

polydopamine-modified mesoporous silica nanoparticles for targeted cancer therapy

Wei Chenga,b,1, Junpeng Nieb,1, Lv Xub,1, Chaoyu Liang a,b, Yunmei Pengb, Gan Liub, Teng Wanga,b, Lin Meib, Laiqiang Huanga,b,*, Xiaowei Zenga,b,*

a

b

Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China The Shenzhen Key Lab of Gene and Antibody Therapy, The Ministry-Province

Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, and Division of Life and Health Sciences, Graduate School at Shenzhen, Tsinghua University , Shenzhen 518055, P.R. China

1

These authors contributed equally to this work.

*

Corresponding author. Tel./Fax: +86 75526036736.

E-mail address: [email protected] (X. Zeng) *

Corresponding author. Tel./Fax: +86 75526036052.

E-mail address: [email protected] (L. Huang)

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 40

ABSTRACT: In this study, we introduced a targeting polymer poly (ethylene glycol)-folic acid (PEG-FA) on the surface of a polydopamine (PDA)-modified mesoporous silica nanoparticles (MSNs) to develop a novel nanoparticle (NP) MSNs@PDA-PEG-FA, which was employed as a drug delivery system loaded with doxorubicin (DOX) as a model drug for cervical cancer therapy. The chemical structure and properties of this NP were characterized by the transmission electron microscopy

(TEM),

X-ray

photoelectron

spectroscopy

(XPS),

N2

adsorption/desorption, dynamic light scattering-autosizer (DLS), thermogravimetric analysis (TGA) and FT-IR spectrophotometer. The pH-sensitive PDA coating served as a gatekeeper. The in vitro drug release experiments showed pH-dependent and sustained drug release profiles which could enhance the therapeutic anti-cancer effect and minimize potential damage to normal cells due to the acidic microenvironment of the tumor. This MSNs@PDA-PEG-FA achieved significantly high targeting efficiency, which was demonstrated by the in vitro cellular uptake and cellular targeting assay. Compared with free DOX and DOX-loaded NPs without folic targeting ligand, the FA targeted NP exhibited higher antitumor efficacy in vivo, implying a highly promising potential carrier for cancer treatments.

KEYWORDS: nanomedicine, mesoporous silica, surface modification, pH-sensitive delivery, cancer targeting

2


Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Today, cancer is one of the greatest public health problems among human beings, and chemotherapy remains to be one of the most common ways used for cancer treatments.1 However, traditional drugs are non-specific and have no ability to discriminate between cancer cells and normal cells, which frequently cause systemic side effects.2 In recent two decades, nanotechnology, especially nanoparticles (NPs) applied in drug delivery systems, have attracted increasing attention. Being incorporated with suitable ligands, NPs used for drug carriers could provide site-specific delivery of anti-cancer agents as well as controlled and sustained drug release.3,4 Besides, owning to enhanced permeability and retention (EPR) effect, NPs possess other advantages including more reasonable biodistribution, high cellular uptake and high encapsulation efficiency.5,6 Among all of the potential alternative supports, mesoporous silica nanoparticles (MSNs) are a promising vector which has been widely studied due to their several unique characteristics like large pore volume and specific surface area, tunable pore structures, high biocompatibility and thermal stability.7-10 Owning to their uniform and large pore size, MSNs have high loading capacity and are able to serve as multifunctional and efficient delivery platforms for various anticancer agents.7,11,12 Moreover, MSNs can be decorated with molecular or polymer moieties on the external surface to make them more controllable in the delivery process.11 Based on these concepts, the outer surface of MSNs can be functionalized with switchable gatekeepers which are sensitive to certain external stimuli, such as pH, light, magnetic 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fields, temperature, antibodies and enzymes, enabling on-command delivery of therapeutic agents.13-15 To enhance the ability of targeting and tumoral uptake, the external surface of NPs is often modified with various cell-interactive ligands (peptides, nucleic acids, antibodies, and small molecules), which can significantly enhance the interaction between the NPs and cancer cells.16,17 However, if the NP surface is not inherently reactive because of the lack of chemically reactive functional groups, the surface decoration can be quite inefficient and cumbersome due to necessary activation of the NP surface with coupling agents or reactive linkers, followed by complicated purification process.18-20 To overcome this challenge, we introduced polydopamine (PDA) into our drug delivery system. PDA is able to form an adhesive layer onto virtually any type of material surfaces and PDA coating has been demonstrated to be a remarkably versatile platform for secondary reactions.16,21 In a weak alkaline condition (pH=~8.5), the catechol group of dopamine is first oxidized to quinone, which then further reacts with other catechols and/or quinones to form a water-insoluble PDA film.16,18,22 Any ligand molecules containing nucleophilic functional groups such as amine or thiol can be immobilized onto the PDA layer via Michael addition and/or Schiff base reactions, allowing the incorporation of different functional ligands on diverse surfaces.16,18,20 To reduce side effects of anticancer drugs to healthy cells and improve the drug efficiency, it is very necessary to improve local effective anticancer drug concentration at specific parts and targeted delivery is a particularly attractive method 4


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to address this challenge.23 In targetable drug delivery systems, the surface of NPs is often properly modified with some small targeting groups like vitamins, antibodies, peptides, and hormones.24,25 Folic acid (FA), a water-soluble B vitamin which is stable and nonimmunogenic, binds selectively to the folate receptor (FR).23,24,26 FR, known as a glycosylphosphatidylinositol (GPI)-linked cell surface receptor, is a high affinity membrane folate-binding glycoprotein.27,28 Since FR is generally overexpressed on the surface of a variety of human cancerous cells and FA has high affinity to FR binding,29-31 a wide range of FA conjugated drug delivery carriers have been largely investigated.32-34 Herein, we introduced polymer poly (ethylene glycol)-folic acid (PEG-FA) to our drug delivery system to fabricate a novel active targeting NP MSNs@PDA-PEG-FA. Besides, PEG-based polymers can help NPs escape from phagocytosis and improve their long-term blood circulation.23,35,36

EXPERIMENTAL SECTION Materials.

Hexadecyl

trimethyl

tetraethylorthosilicate (TEOS), dopamine

ammonium

bromide

(CTAB),

hydrochloride, mercapto group-terminated

poly(ethylene glycol) (PEG-SH), mercapto group-terminated poly (ethylene glycol)-folic

acid

(SH-PEG-FA),

methanol,

acetonitrile,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and 4',6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin hydrochloride (DOX) was purchased from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ammonium fluoride (NH4F) was bought from Aladdin Industrial Co., Ltd. (Shanghai, China). Human cervix carcinoma cell line HeLa was purchased from American Type Culture Collection (ATCC, Rockville, MD). Preparation of MSNs. For the synthesis of MSNs, a modified procedure was used as previously described.37 CTAB (1.82 g, 5 mmol) and NH4F (3 g, 81 mmol) were dissolved in turn in 500mL of deionized water, followed by heating up the solution to 80℃ under intensive stirring. Thereafter, TEOS (9 mL, 8.41 g) was added dropwise to the above surfactant solution in sequence. The mixture solution was allowed to vigorously stir in an oil bath at 80℃ for another 6 h. The resulting white precipitates (as-MSNs) were collected by centrifugation (12000 rpm, 10 min) and washed with DI water and ethanol repeatedly. Then, the product was dried under vacuum at 40℃ overnight. The surfactant template (CTAB) was removed by calcination. The as-MSNs were first heated to 300℃ at a rising rate of 2℃·min-1 and heated to 600℃ at a rising rate of 1℃·min-1. Then the product was kept at 600℃ for 6 h. Loading drug into MSNs. Doxorubicin hydrochloride (DOX) was loaded into MSNs through diffusion.38 MSNs (100 mg) were suspended in 10 mL of aqueous solution. Then doxorubicin hydrochloride (100 mg) was dissolved in the mixture and stirred for 24 h in dark at room temperature. After that the products were collected by centrifugation and washed with deionized water to remove the excess DOX. Subsequently, DOX-loaded MSNs were lyophilized and designated as MSNs-DOX. Prime-coating with PDA. The core particles were coated with PDA as follows: 6


Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a total of 100 mg particles were suspended in 50 mL dopamine hydrochloride solution (1 mg mL-1) in Tris buffer (pH 8.5, 10 mM) and the reaction was taken at room temperature under vigorous stirring for 6 h. The obtained black particles were centrifuged and washed with distilled water. The PDA-coated nanoparticles were then dried by lyophilization. Conjugation of PEG or PEG-FA to PDA-coated MSNs. The functional ligands were bound to the surface of PDA-coated MSNs via Michael addition reaction. In brief, a 100 mg amount of PDA-coated nanoparticles were well resuspended in 20 mL Tris-HCl buffer (pH 8.5, 10mM) containing 100 mg of different ligands (PEG-SH or FA-PEG-SH), followed by the addition of 2 mg TCEP. After 6 h of vigorous stirring at room temperature, the resulting particles (designated as MSNs-DOX@PDA-PEG and MSNs-DOX@PDA-PEG-FA) were collected by centrifugation and washed with distilled water to remove residual reactants. The final products were then lyophilized. Characterization of nanoparticles. The particle size and zeta potential measurements of the prepared NPs were carried out on Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). The dried powder samples were diluted with distilled water and sonicated before measurement. The shape and surface morphology of the NPs were imaged by a transmission electron microscopy (TEM, Tecnai G2 F30, FEI Company, Hillsboro, Oregon, USA). To prepare samples for TEM, appropriate amount of particles were suspended in distilled water. After sonication, a drop of sample suspensions was deposited onto a carbon-coated copper grid and dried overnight before observation. N2 adsorption–desorption isotherms were recorded at 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

-196 ℃ using an ASAP 2020 accelerated surface area and porosity analyzer (Micromeritics, USA). The samples were degassed in a vacuum at 120 °C for 24 h before the measurements were conducted. Specific surface areas of the samples were determined from the adsorption data following the Brunauer–Emmett–Teller (BET) method. The total pore volumes and pore size were estimated using the Barrett– Joyner–Halenda (BJH) model. X-ray photoelectron spectroscopy (XPS) was performed on an AXIS His XPS spectrometer (Kratos Ltd., UK). The existence of certain elements on the surface of the NPs was confirmed according to specific binding energy (eV),39 which ranged from 0 to 1300 eV, using a monochromatic Al Kα X-ray source (1486.6 eV photons, 150 W). The IR spectra of the samples was obtained on a FT-IR spectrophotometer (Thermo Nicolet, Madison, Wisconsin) in the range of 4000 and 500 cm-1. A thermal gravimetric analyzer (Netzsch STA 449, Germany) was employed for the thermogravimetric analysis (TGA) of the NPs. The samples were heated to 800℃ under a heating ramp of 10℃ per minute. Drug loading content (LC) of DOX-loaded NPs were determined by HPLC (LC 1200, Agilent Technologies, USA) using previously published methods.37 The HPLC analysis of DOX was achieved on a reverse-phase C-18 column (150 × 4.6 mm, 5µm, C18, Agilent Technologies, CA, USA) with a mobile phase consisting of phosphate buffer (25 mmol L-1 Na2HPO4 30 mmol L-1 NaH2PO4, pH 5.0), methanol and acetonitrile (30:50:20, v/v/v). The flow rate of mobile phase was set at 1 ml min-1. The column effluents were monitored using a UV detector at 233 nm. In the preparation process of the NPs, all the supernatants and washings were collected and 8


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


combined. The concentration of the remaining DOX in the solution could be calculated through a calibration curve. The LC of the DOX-loaded NPs was calculated on the basis of the following equation.

LC% =

weight of DOX in the nanoparticles × 100% weight of nanoparticles

In vitro drug release profiles. To investigate in vitro DOX release, a slightly modified method was used.40 Briefly, 5 mg of accurately weighed lyophilized samples was dispersed in I mL of phosphate buffer solution (PBS pH 7.4 5.6 2.0) and then transferred into a dialysis bag (MWCO=3,500, Shanghai Sangon, China) with the outer centrifuge tube filled with 10 mL of similar release medium. The tube was placed in an orbital shaker water bath and shaken at 37℃ at 90 rpm. The whole release buffer outside the dialysis bag was collected and replaced with equal volumes of fresh buffer at predetermined time intervals for 7 days. The amount of DOX in the supernatant was evaluated by HPLC. Cell culture and in vitro cellular uptake. Hela cells were routinely cultured in Dulbecco’s Modified Eagle medium (DMEM) media supplemented with 10% (v/v) fetal bovine serum (FBS), antibiotics penicillin (100 IU mL-1) and streptomycin (100 µg mL-1). Cells were cultivated under a humidified atmosphere with 5% CO2 – 95% air atmosphere at 37℃. The cellular uptake was investigated by treating the cells with free DOX or DOX-loaded NPs. Briefly, the cells were treated with either free DOX or NPs 9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(equivalent to 5 µg mL-1 of DOX) in a 20 mm glass-bottom Petri dish for 0.5 or 2 h. After incubation, the cells were then washed three times in PBS buffer and stained with DAPI for another 20 min. After that, the cells were washed with cold PBS three times to remove free DAPI and visualized under a confocal laser scanning microscopy (CLSM, Olympus Fluoview FV-1000, Tokyo, Japan) with the following channels: red channel (DOX) excited at 488 nm, blue channel (DAPI) excited at 358 nm. The quantitative analysis was performed by flow cytometry. Hela cells were seeded into 6-well plates at the density of 1×105 cells/well and allowed to attach overnight. After incubation with free DOX (5 µg mL-1) and NPs (equivalent DOX concentration with the free DOX group) for 2 h at 37℃, the untouched DOX and NPs were removed by sufficient PBS washing. Then the cells were trypsinized and collected by centrifugation to obtain a cell pellet. Finally the cells were suspended in PBS buffer and observed by a flow cytometer (BD Biosciences, San Jose, CA, USA) at excitation wavelength of 488 nm and emission wavelength of 590 nm. In vitro cytotoxicity. NPs antitumor activity against Hela cells was evaluated by the MTT assay in vitro. Hela cells were seeded in 96-well culture plates (8×103 cells per well). After incubation overnight, the cells were exposed to different concentrations of free DOX or DOX-loaded NPs (ranging from 0.125 to 2.5 µg mL-1 DOX equivalent concentration) for 24 h, 48 h, 72 h, and then 10 µL of MTT (5 mg mL-1) stock solution was added to each well. The incubation continued for another 4 h. Next, the medium was removed and the resulting formazan crystals formed in living cells were solubilized by DMSO (100 µL/well). After gentle shake for 10 min, the 10


Page 10 of 40

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


absorbance was analyzed on a microplate reader (Bio-Rad Model 680, UK) at a wavelength of 490 nm. To normalize absorbance values, the value of cells in medium without drugs was assigned to 1 and the value of cells without addition of MTT was designated as 0. Xenograft tumor models. All animal experiment procedures were conducted with the approval of the Administrative Committee on Animal Research in the Tsinghua University. Five to six weeks old female athymic nude mice were obtained from the Guangdong Medical Laboratory Animal Center and housed under a sterile condition. After acclimatization for 1 week, each mouse received a subcutaneous injection of 2×106 Hela cells in PBS (100 µL) in the flank of right fore leg. The tumor volumes were monitored at predetermined intervals using a digital caliper. Tumor volume (V) was calculated using the formula: V = d2 × D/2, where d is the short axis and D is the long axis.41 In vivo imaging and biodistribution analysis. When the tumor size reached an average volume of around 80 mm3, the mice were randomly divided into three groups, each group received (1) free DOX (4 mg kg-1), (2) MSNs-DOX@PDA-PEG, and (3) MSNs-DOX@PDA-PEG-FA (4 mg kg-1 equivalent DOX for NPs) via tails, respectively. The mice were humanely sacrificed at 3 and 24 h respectively after treatment, and the organs (i.e. heart, liver, spleen, lung and kidney) and tumors were collected without delay. The fluorescence intensities of DOX in all organs and tumors were measured with the Maestro™ Automated In-Vivo Imaging system (CRi Maestro™, USA). 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 40

In vivo antitumor efficacy. To evaluate the in vivo antitumor efficacy of the NPs, Hela tumor-bearing mice were prepared as mentioned above. When the tumor volume reached approximately 80 mm3, the mice were randomly divided into five groups (each group has five animals): (1) saline group, (2) free DOX (5 mg DOX kg-1) group, (3)

MSNs-DOX@PDA-PEG

(5

mg

DOX

kg-1)

group,

(4)

MSNs-DOX@PDA-PEG-FA (5 mg DOX kg-1) group, and (5) drug free MSNs@PDA-PEG-FA.

The saline group was used as a reference and the drug free

MSNs @PDA-PEG-FA group was taken as a negative control. Inoculation was performed by an intraperitoneal injection of relevant sample in 100 µl PBS on Day 0, 4, 8 and 12. Tumor size and body weights were measured every two days. The mice were euthanized on Day 16. The anti-tumor activity was evaluated via tumor growth and terminal tumor weight.42 Statistical analysis. Unless stated otherwise, all the experiments were carried out at least three times. The experimental data are expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA followed by Bonferroni test with SPSS 22.0 software. *P < 0.05 as statistical significance and **P < 0.01 as extreme statistical significance.

RESULTS AND DISCUSSIONS Synthesis of MSNs-DOX@PDA-PEG-FA. The design and synthetic strategy of MSNs-DOX@PDA-PEG-FA nanoparticles is illustrated in Scheme 1 and the procedure can be divided into four steps. Firstly, an MSN material was synthesized 12


Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


according to a previously reported method.37 The removal of CTAB which acted as the surfactants and mesostructural template resulted in a dramatic increase of pore volume and specific surface area. Then the DOX was loaded into MSNs via diffusion in an aqueous media. Thereafter, PDA-coated NPs were synthesized using a promising method based on the oxidative self-polymerization of dopamine following a literature protocol.42 The solution turned dark when reaction finished, which indicated that dopamine was successfully polymerized. Finally, we used a folic acid conjugated PEG (PEG-FA) to functionalize the NPs in order to enhance the targeting effect and improve the long-term blood circulation. Characterization of nanoparticles. The particle size and size distribution of DOX-loaded NPs are detailed in Table 1. As shown in Table 1, the fabricated NPs exhibited hydrodynamic sizes around 140-190 nm in diameter, which is theoretically suitable for high cellular uptake and penetration of NPs into tumors due to the EPR effect.39,43 MSNs-DOX NP has an average size of 153.5 ± 6.7 nm, while that of MSNs-DOX@PDA was 182.2 ± 2.2 nm and that of MSNs-DOX@PDA-PEG-FA was 193.08

±

8.1

nm.

Comparing

with

MSNs-DOX,

the

particle

size

of

MSNs-DOX@PDA increased by approximately 30 nm, which could be regarded as an evidence of the successful coating of PDA. Furthermore, the conjugation of PEG-FA did not led to a significant increase in the particle size. The polydispersity index (PDI) of all five NPs was in the range of 0.153-0.276, which was an acceptable size distribution. Zeta potential plays a key role for the stability of nanoparticles in suspension 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

through the electrostatic repulsion between particles as well as the interaction between the nanoparticles and the tumor cell membrane.5,44 The zeta potential values are displayed in Table 1. The mean surface charge of MSNs was -15.7 ± 0.6 mV. The negative zeta potential of bare MSNs may be ascribed to the presence of high amount of silanol groups on the silica surface.45,46 When the MSNs were loaded with DOX, the zeta potential increased to -7.2 ± 0.4 mV, implying that the positively charged amino groups on DOX neutralized partial negative charge of the silanol groups. The surface modification with PDA lowered the value to -9.8 ± 0.3 mV. The change in the value may be ascribed to the deprotonation of phenolic hydroxyl groups of polydopamine at neutral pH.47 After functionalization with PEG-FA, the zeta potential of NPs was also negative (-4.8 ± 0.9). This result could be due to the carboxylic acid in FA structure.45 Slightly negative charge would be appropriate for both cell accessibility and NPs dispersibility.48 The

morphology of DOX-loaded

MSNs,

MSNs @PDA and MSNs

@PDA-PEG-FA was verified by TEM. Images of these NPs are compared in Figure 1 (A)-(C). All of the NPs own a uniform and monodispersed spherical shape and a highly porous structure. The diameters of the periodical, well-organized hexagonal mesopores measured from the images are around 2-3 nm. For the micrographs of MSNs-DOX@PDA and MSNs-DOX@PDA-PEG-FA, an easily identified rough shell was observed, which is an evidence of the formation of the PDA films. The average particle size of NPs obtained from TEM is about 130-180 nm in diameter, which is a similar to that measured by DLS experiments. 14


Page 14 of 40

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The mesoporous features of the NPs were further confirmed by N2 adsorption-desorption. The pore volume, specific surface area, and the most probable pore size are listed in Table 2 and the corresponding BJH pore size distribution of the NPs are shown in Figure 1 (E). For the bare MSNs, the BET surface area was 159.49 m2g-1, the pore volume was 0.5 cm3g-1 and the pore size estimated by BJH method was about 2.56 nm. After loading drug, MSNs-DOX showed a decrease in the specific surface area (141.77 m2g-1), pore volume (0.42 cm3g-1) and the pore size (2.32 nm). The values of MSNs-DOX@PDA decreased further, which were 79.33 m2g-1, 0.34 cm3g-1 and the 2.07 nm, respectively. These results demonstrate that DOX occupied the mesoporous channels of silica and PDA obstructed the entrance of the channels. To determine the surface composition of the NPs, XPS was studied and presented in Figure 2. Compared with bare MSNs, the appearance of N1s spectrum at the binding energy of ∼400 eV of MSNs @PDA and MSNs @PDA-PEG-FA illustrated the presence of a PDA layer.49 As for silicon peaks (Si2p), only MSNs contains silicon atoms in its chemical structure, whereas PDA, PEG and FA do not, therefore the lower intensity of Si2p (MSNs@PDA or MSNs@PDA-PEG-FA v.s. bare MSNs) at ∼104 eV could also confirm the existence of PDA films. The intensity of Si2p peak for MSNs@PDA-PEG-FA is slightly smaller than that for MSNs@PDA, which evidences the incorporation of ligand PEG-FA. Furthermore, the C1s region can also be used to confirm the PDA coating or folate incorporated onto the nanospheres (Figure S2). The existence of a low carbon concentration of bare MSNs may be attributed to the surface adventitious carbon from the residual carbon of the 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

organic template or environment contamination. After PDA modification, a large C1s peak at 284.8 eV can be detected, which is contributed by C-C/C-O groups in PDA. In addition, the intensity of C1s peak in MSNs@PDA-PEG-FA was more expressive than that in MSNs@PDA due to the increase of carbon atom ratio in the ligand PEG-FA. The surface characterization of prepared NPs was further evaluated using FT-IR. The FT-IR spectra are plotted in Figure 3 (A). The spectra of all samples displayed peaks at 1050 cm-1 and 967 cm-1, which were Si-O-Si stretching vibration and silanol group vibration, respectively.50 After coating the polydopamine, several new absorption signals appeared. The peak appearing at 1635 cm-1 was attributed to the aromatic rings skeleton stretching vibration51 and the broad absorbance located at 3423 cm-1 was assigned to the stretching vibrations of N-H/O-H,52 which supported the PDA coating on the surface of MSNs. As for MSNs@PDA-PEG-FA, the peaks at 1493 cm-1 and 1445 cm-1 indicating the existence of targeted ligand FA onto the NPs. Further, TGA was performed and the curve was shown in Figure 3 (B). The weight loss of bare MSNs was7.08wt%. Nevertheless, after surface functionalization of MSNs, the weight loss increased to 17.73wt% for MSNs@PDA and 27.35wt% for MSNs@PDA-PEG-FA, respectively, indicating that about 10.65wt% of PDA and 9.62wt% of PEG-FA were introduced onto the surface of MSNs. This result again demonstrates that the objective functional groups were successfully functionalized on the surfaces of NPs. In vitro drug release kinetics. The capability of controllable drug release of the 16


Page 16 of 40

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NPs and their pH dependency were investigated at 37℃ under pH 7.4, 5.6 and 2.0 PBS solutions. As shown in Figure 4, all of the NPs showed a typically biphasic release pattern: an initial burst release of DOX in the first day followed by a sustained drug release up to 190 h. Under all test pH values, the MSNs-DOX exhibited a higher DOX release rate than MSNs-DOX@PDA and MSNs-DOX@PDA-PEG-FA. The distinct release profile suggested that the PDA films coated on the surfaces of the NPs could block the pores of MSNs and effectively suppress drug release. For the PDA-coated

NPs

(MSNs-DOX@PDA and

MSNs-DOX@PDA-PEG-FA),

an

increasing drug release rate can be observed as the acidity of the solution increased. At

pH

7.4,

the

cumulative

release

for

MSNs-DOX@PDA

and

MSNs-DOX@PDA-PEG-FA was 27.9% and 28.5% respectively over the course of 190 h, whereas the PDA-loaded NPs exhibited higher release content with 37.2% at pH 5.6 and up to 51.1% at pH 2.0 for MSNs-DOX@PDA and with 38.3% and 49.5% at pH 5.6 and at pH 2.0 for MSNs-DOX@PDA-PEG-FA. The result indicated that at physiological pH, the PDA-coated aggregates could basically preserve the structures of the drug delivery system, providing slow drug diffusion and durable release over a long period. In comparison, when PDA-modified NPs were dispersed in acidic conditions, the loaded-DOX rapidly released due to the brakeage of PDA films, which unlocked the channel of the NPs. Owing to the acidic microenvironment of the tumor and intracellular acidic endosomes and lysosomes,53 the pH-dependent release behavior can enhance the therapeutic anti-cancer effect as well as minimize potential damage to normal cells.8,54 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 40

For non-PDA-coated NPs (MSNs-DOX), however, we found that as the increasing of acidity, the DOX release rate was also noticeably increased. This could be explained by the acidity-induced dissolution of DOX in aqueous environments. Acidic conditions could enhance the solubility of DOX,55 resulting in a faster drug release. In vitro cellular uptake. The extent of the internalization of DOX and DOX-loaded NPs into Hela cells was investigated qualitatively by confocal microscopy. It can be seen from Figure 5 (A), a week intensity of fluorescence could be detected after incubation for 30 min, suggesting that the NPs and free DOX can enter Hela cells in as short as 0.5 h. Furthermore, it could be observed from Figure 5 (B)

that

the

fluorescent

intensity

in

Hela

cells

incubated

with

MSNs-DOX@PDA-PEG-FA for 2 h was higher than that of the cells incubated with MSNs-DOX@PDA-PEG, indicating a higher level of cellular uptake due to the incorporation of folate onto the surface of the NPs. To further investigate the important role of folic acid in the cellular uptake of MSNs-DOX@PDA-PEG-FA, we conducted a receptor competition assay by selecting folic acid as the competitive reagent. Consisted with our hypothesis, when MSNs-DOX@PDA-PEG-FA and folic acid were added to wells simultaneously, the fluorescent intensity significantly decreased. The endocytosis of DOX and DOX-loaded NPs was further confirmed by flow cytometry results (Figure 5 (C) and (D)). Quantitative analysis indicated that MSNs-DOX@PDA-PEG-FA exhibited 1.8-fold higher uptake by Hela cells than did MSNs-DOX@PDA-PEG, reinforcing that FA can enhance the endocytosis of NPs 18


Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


through the specific interactions with cervical cancer cells. In vitro cellular cytotoxicity. The cell viability of Hela cells was assessed using MTT to investigate the cytotoxicity of DOX-loaded NPs. The cytotoxicity of

the

drug-free nanoparticles to Hela cells was also evaluated to eliminate any non-specific effect. As shown in Figure 6, the viability of Hela cells remained above 82% even after incubation with 250 µ g mL-1 MSNs or MSNs@PDA-PEG-FA nanoparticles respectively for 72 h. This clearly indicated that the synthesized MSNs and MSNs@PDA-PEG-FA nanoparticles were biocompatible and produced no significant toxicity to tissues and cells. As can be seen in Figure 6, MSNs-DOX@PDA-PEG-FA exhibited better in vitro inhibition effect of cell growth than MSNs-DOX@PDA and MSNs-DOX@PDA-PEG. For example, the Hela cellular viability (48 h, 1 µ g mL-1) was 33.4% for MSNs-DOX@PDA, 29.6% for MSNs-DOX@PDA-PEG and 20.7% for MSNs-DOX@PDA-PEG-FA. Furthermore, the cell viability decreased as the concentration and incubation time increased, suggesting a concentration-dependent and time-dependent cytotoxic effect. Interestingly, we found that clinical available DOX displayed better In vitro anti-tumor efficacy than MSNs-DOX@PDA-PEG-FA. Similar results could also be found in some previous studies.56 However, free DOX could cause severe side effects such as cardiotoxicity and metabolism by liver. By contrast, DOX-loaded NPs can enhance the in vivo biodistribution, bioavailability and also effectively reduce the side effect of chemotherapeutic agents owning to the sustained release of anticancer drugs and active targeting effect. MSNs-DOX also showed a high cellular cytotoxicity, 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40

which was attributed to the much faster drug release than PDA-coated NPs. In vivo biodistribution study. To evaluate the in vivo distribution of DOX and tumor targeting of MSNs-DOX@PDA-PEG-FA, the tumor-bearing mice were intravenously

injected

with

free

DOX,

MSNs-DOX@PDA-PEG

and

MSNs-DOX@PDA-PEG-FA via tails. The distribution of DOX can be directly tracked through ex vivo fluorescence imaging of organs owning to the intrinsic fluorescence of DOX. As shown in Figure 7, a strong fluorescence signal could be detected at the tumor issue for all groups at 3 h post-injection. The tumor fluorescence intensity of MSNs-DOX@PDA-PEG-FA was stronger than that of free DOX and MSNs-DOX@PDA-PEG. After 24 h post-injection, the signal intensity of liver and kidney began to wane, while the signal in tumor tissues increased, especially for MSNs-DOX@PDA-PEG and MSNs-DOX@PDA-PEG-FA groups due to the long-term circulation of PEG-based NPs. In addition, the tumor signal intensity of MSNs-DOX@PDA-PEG-FA was observed significantly stronger than that of MSNs-DOX@PDA-PEG, indicating that MSNs-DOX@PDA-PEG-FA exhibited the most remarkable in vivo tumor targeting effect. In vivo antitumor effects. To investigate in vivo antitumor activity, Hela tumor-bearing nude model mice were prepared. Tumor volume and body weight of the mice were measured for 16 days. As can be seen in Figure 8 (B), compared with saline-treated group and MSNs@PDA-PEG-FA-treated group, both DOX-loaded NPs and free DOX treatment significantly suppressed tumor growth. However, the two DOX-loaded NPs exhibited higher antitumor effect than did free DOX, probably 20


Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


attributing to the EPR effect of the NPs. In the meantime, that of targeted MSNs-DOX@PDA-PEG-FA

was

superior

to

that

of

non-targeted

MSNs-DOX@PDA-PEG, which was due to the active targeting effect of FA-modified NPs to tumor cells, allowing a relatively higher level of drug concentration at the tumor site and further improving drug efficacy. Furthermore, no marked body weight change was observed for NPs-treated groups (Figure 8 (D)), indicating that the synthesized NPs have good biocompatibility. The tumor morphology and average weight of each group are shown in Figure 8 (A) and Figure 8(C), directly demonstrating

the

excellent

tumor-inhibiting

effect

of

the

targeted

MSNs-DOX@PDA-PEG-FA. Histological analysis. To further assess the in vivo therapy efficacy and toxicity of the NPs, histology analysis was employed (Figure 9). Major organs such as heart, liver, spleen, lung, kidney and tumor of mice were collected and sectioned for hematoxylin and eosin (H&E) staining. From Figure 9, we can see that after treated with DOX or DOX loaded NPs, especially MSNs-DOX@PDA-PEG-FA, most of tumor tissue cells were destroyed and became necrotic, while the cells largely or partially retain their normal morphology for the control and MSNs@PDA-PEG-FA treated groups. Moreover, no noticeable tissue damage could be found in the other organs in any of the treatment groups for the test dose. These results indicate the promising in vivo application of this MSNs@PDA-PEG-FA theranostic delivery platform.

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

CONCLUSIONS In this study, we successfully synthesized a novel system of folic acid-functionalized

polydopamine-modified

NPs

(MSNs@PDA-PEG-FA)

for

sustained and controlled delivery of DOX for targeted cervical cancer therapy. MSNs-DOX@PDA-PEG-FA had a hydrodynamic size of about 200 nm in diameter and high drug loading content. The PDA film on the surface of the NPs was clearly observed via TEM and the successful conjugation of PEG-FA was demonstrated by FT-IR

and

XPS.

The

in

vitro

drug

release

profiles

indicated

that

MSNs-DOX@PDA-PEG-FA was strongly sensitive to pH. The in vitro cellular uptake and cellular targeting assays demonstrated that MSNs-DOX@PDA-PEG-FA managed to target Hela cells specifically with higher efficacy than MSNs-DOX@PDA-PEG. MSNs-DOX@PDA-PEG-FA had high cytotoxicity and blank MSNs @PDA-PEG-FA was biocompatible and essentially nontoxic. The in vitro and in vivo experiments demonstrated that the MSNs-DOX@PDA-PEG-FA had enhanced therapeutic effects and exhibited superior antitumor effects. It is the first time to report the excellent advantages of such MSNs-DOX@PDA-PEG-FA NP in the literature, which is a promising nanocarrier of different antitumor drugs for cancer treatment.

ASSOCIATED CONTENT Supporting information. Size distribution of MSNs and MSNs-DOX@PDA-PEG-FA. XPS spectra of narrow scan for C1s peaks of MSNs, MSNs@PDA and MSNs@PDA-PEG-FA. The 22


Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


supporting

information

is

available

free of

charge

via

the Internet

at

http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail

address: [email protected] (X. Zeng)

*E-mail

address: [email protected] (L. Huang)

Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We are grateful for the financial support from the National Natural Science Foundation of China (No. 31270019), the Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306036), the Natural Science Foundation of Guangdong Province (No. 2015A030313848), Guangdong Special Support Program, Science and Technology Planning Project of Guangdong Province (No. 2016A020217001), Science, Technology & Innovation Commission of Shenzhen Municipality

(Nos.

JCYJ20160531195129079,

JCYJ20160301152300347,

JCYJ20160429182415013 and JCYJ20150430163009479).

REFERENCES (1) Xiao, D.; Jia, H. Z.; Ma, N.; Zhuo, R. X.; Zhang, X. Z. A Redox-Responsive Mesoporous Silica Nanoparticle Capped with Amphiphilic Peptides by Self-Assembly for Cancer Targeting Drug Delivery. Nanoscale 2015, 7, 10071-10077. (2) Timko, B. P.; Dvir, T.; Kohane, D. S. Remotely Triggerable Drug Delivery Systems. Adv. Mater. 2010, 22, 4925-4943. (3) Gallo, J.; Long, N. J.; Aboagye, E. O. Magnetic Nanoparticles as Contrast Agents in the Diagnosis and Treatment of Cancer. Chem. Soc. Rev. 2013, 42, 7816-7833. 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 40

(4) Mei-Chin, C.; Sonaje, K.; Ko-Jie, C.; Hsing-Wen, S. A Review of the Prospects for Polymeric Nanoparticle Platforms in Oral Insulin Delivery. Biomaterials 2011, 32, 9826-9838. (5) Zeng, X.; Tao, W.; Mei, L.; Huang, L.; Tan, C.; Feng, S. S. Cholic Acid-Functionalized Nanoparticles of Star-Shaped PLGA-vitamin E TPGS Copolymer for Docetaxel Delivery to Cervical Cancer. Biomaterials 2013, 34, 6058-6067. (6) Tomasina, J.; Lheureux, S.; Gauduchon, P.; Rault, S.; Malzert-Fréon, A. Nanocarriers for the Targeted Treatment of Ovarian Cancers. Biomaterials 2013, 34, 1073-1101. (7) Chan, M. H.; Lin, H. M. Preparation and Identification of Multifunctional Mesoporous Silica Nanoparticles for in Vitro and in Vivo Dual-Mode Imaging, Theranostics, and Targeted Tracking. Biomaterials 2015, 46, 149-158. (8) Wang, Y.; Shi, W.; Wang, S. S.; Li, C. Y.; Qian, M.; Chen, J.; Huang, R. Q. Facile Incorporation of Dispersed Fluorescent Carbon Nanodots Into Mesoporous Silica Nanosphere for pH-triggered Drug Delivery and Imaging. Carbon 2016, 108, 146-153. (9) Rosenholm, J. M.; Mamaeva, V.; Sahlgren, C.; Linden, M. Nanoparticles in Targeted Cancer Therapy: Mesoporous Silica Nanoparticles Entering Preclinical Development Stage. Nanomedicine-UK 2012, 7, 111-120. (10) Li, Q.; Xu, S.; Zhou, H.; Wang, X.; Dong, B.; Gao, H.; Tang, J.; Yang, Y. PH and Glutathione Dual-Responsive

Dynamic

Cross-Linked

Supramolecular

Network

on

Mesoporous

Silica

Nanoparticles for Controlled Anticancer Drug Release. ACS Appl. Mater. Interfaces 2015, 7, 28656-28664. (11) Li, Z. X.; Barnes, J. C.; Bosoy, A.; Stoddart, J. F.; Zink, J. I. Mesoporous Silica Nanoparticles in Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2590-2605. (12) Li, T.; Shen, X.; Geng, Y.; Chen, Z.; Li, L.; Li, S.; Yang, H.; Wu, C.; Zeng, H.; Liu, Y. Folate-Functionalized Magnetic-Mesoporous Silica Nanoparticles for Drug/Gene Codelivery to Potentiate the Antitumor Efficacy. ACS Appl. Mater. Interfaces 2016, 8, 13748-13758. (13) Gimenez, C.; de la Torre, C.; Gorbe, M.; Aznar, E.; Sancenon, F.; Murguia, J. R.; Martinez-Manez, R.; Marcos, M. D.; Amoros, P. Gated Mesoporous Silica Nanoparticles for the Controlled Delivery of Drugs in Cancer Cells. Langmuir 2015, 31, 3753-3762. (14) Xu, X. B.; Lu, S. Y.; Gao, C. M.; Feng, C.; Wu, C.; Bai, X.; Gao, N. N.; Wang, Z. Y.; Liu, M. Z. Self-Fluorescent and Stimuli-Responsive Mesoporous Silica Nanoparticles Using a Double-Role Curcumin Gatekeeper for Drug Delivery. Chem. Eng. J. 2016, 300, 185-192. (15) Coll, C.; Bernardos, A.; Martinez-Manez, R.; Sancenon, F. Gated Silica Mesoporous Supports for Controlled Release and Signaling Applications. Accounts Chem. Res. 2013, 46, 339-349. (16) Gullotti, E.; Park, J.; Yeo, Y. Polydopamine-Based Surface Modification for the Development of Peritumorally Activatable Nanoparticles. Pharm. Res.-Dordr. 2013, 30, 1956-1967. (17) Peer, D.; Karp, J. M.; Hong, S.; FaroKhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751-760. (18) Park, J.; Brust, T. F.; Lee, H. J.; Lee, S. C.; Watts, V. J.; Yeo, Y. Polydopamine-Based Simple and Versatile Surface Modification of Polymeric Nano Drug Carriers. ACS Nano 2014, 8, 3347-3356. (19) Cheng, J.; Teply, B. A.; Sherifi, I.; Sung, J.; Luther, G.; Gu, F. X.; Levy-Nissenbaum, E.; Radovic-Moreno, A. F.; Langer, R.; Farokhzad, O. C. Formulation of Functionalized PLGA-PEG Nanoparticles for in Vivo Targeted Drug Delivery. Biomaterials 2007, 28, 869-876. (20) Zhu, D. W.; Tao, W.; Zhang, H. L.; Liu, G.; Wang, T.; Zhang, L. H.; Zeng, X. W.; Mei, L. Docetaxel (DTX)-loaded Polydopamine-Modified TPGS-PLA Nanoparticles as a Targeted Drug 24


Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Delivery System Fore the Treatment of Liver Cancer. Acta Biomater. 2016, 30, 144-154. (21) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426-430. (22) Lee, H.; Rho, J.; Messersmith, P. B. Facile Conjugation of Biomolecules Onto Surfaces Via Mussel Adhesive Protein Inspired Coatings. Adv. Mater. 2009, 21, 431. (23) Tao, W.; Zhang, J. X.; Zeng, X. W.; Liu, D.; Liu, G.; Zhu, X.; Liu, Y. L.; Yu, Q. T.; Huang, L. Q.; Mei, L. Blended Nanoparticle System Based on Miscible Structurally Similar Polymers: A Safe, Simple, Targeted, and Surprisingly High Efficiency Vehicle for Cancer Therapy. Adv. Healthc. Mater. 2015, 4, 1203-1214. (24) Salmaso, S.; Semenzato, A.; Caliceti, P.; Hoebeke, J.; Sonvico, F.; Dubernet, C.; Couvreur, P. Specific Antitumor Targetable Beta-Cyclodextrin-Poly(Ethylene Glycol)-Folic Acid Drug Delivery Bioconjugate. Bioconjugate Chem. 2004, 15, 997-1004. (25) Jin, C. S.; Cui, L. Y.; Wang, F.; Chen, J.; Zheng, G. Targeting-Triggered Porphysome Nanostructure Disruption for Activatable Photodynamic Therapy. Adv. Healthc. Mater. 2014, 3, 1240-1249. (26) Chowdhuri, A. R.; Singh, T.; Ghosh, S. K.; Sahu, S. K. Carbon Dots Embedded Magnetic Nanoparticles @Chitosan @Metal Organic Framework as a Nanoprobe for pH Sensitive Targeted Anticancer Drug Delivery. ACS Appl. Mater. Interfaces 2016, 8, 16573-16583. (27) Sudimack, J.; Lee, R. J. Targeted Drug Delivery Via the Folate Receptor. Adv. Drug Delivery Rev. 2000, 41, 147-162. (28) Porta, F.; Lamers, G. E. M.; Morrhayim, J.; Chatzopoulou, A.; Schaaf, M.; den Dulk, H.; Backendorf, C.; Zink, J. I.; Kros, A. Folic Acid-Modified Mesoporous Silica Nanoparticles for Cellular and Nuclear Targeted Drug Delivery. Adv. Healthc. Mater. 2013, 2, 281-286. (29) Karamipour, S.; Sadjadi, M. S.; Farhadyar, N. Fabrication and Spectroscopic Studies of Folic Acid-Conjugated Fe3O4@Au Core-Shell for Targeted Drug Delivery Application. Spectrochim. Acta a 2015, 148, 146-155. (30) Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.; Leamon, C. P. Folate Receptor Expression in Carcinomas and Normal Tissues Determined by a Quantitative Radioligand Binding Assay. Anal. Biochem. 2005, 338, 284-293. (31) Leamon, C. P.; Reddy, J. A. Folate-Targeted Chemotherapy. Adv. Drug Delivery Rev. 2004, 56, 1127-1141. (32) Zhu, Y. F.; Fang, Y.; Kaskel, S. Folate-Conjugated Fe3O4@SiO2 Hollow Mesoporous Spheres for Targeted Anticancer Drug Delivery. J. Phys. Chem. C 2010, 114, 16382-16388. (33) Zhang, X. K.; Meng, L. J.; Lu, Q. H.; Fei, Z. F.; Dyson, P. J. Targeted Delivery and Controlled Release of Doxorubicin to Cancer Cells Using Modified Single Wall Carbon Nanotubes. Biomaterials 2009, 30, 6041-6047. (34) Stella, B.; Arpicco, S.; Peracchia, M. T.; Desmaele, D.; Hoebeke, J.; Renoir, M.; D'Angelo, J.; Cattel, L.; Couvreur, P. Design of Folic Acid-Conjugated Nanoparticles for Drug Targeting. J. Pharm. Sci.-US 2000, 89, 1452-1464. (35) Shi, J. J.; Xiao, Z. Y.; Votruba, A. R.; Vilos, C.; Farokhzad, O. C. Differentially Charged Hollow Core/Shell Lipid-Polymer-Lipid Hybrid Nanoparticles for Small Interfering RNA Delivery. Angew. Chem. Int. Edit. 2011, 50, 7027-7031. (36) Liu, G.; Gao, H.; Zuo, Y.; Zeng, X.; Tao, W.; Tsai, H.; Mei, L. DACHPt-Loaded Unimolecular Micelles Based on Hydrophilic Dendritic Block Copolymers for Enhanced Therapy of Lung Cancer. 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Appl. Mater. Interfaces 2017, 9, 112-119. (37) Chang, D.; Gao, Y.; Wang, L.; Liu, G.; Chen, Y.; Wang, T.; Tao, W.; Mei, L.; Huang, L.; Zeng, X. Polydopamine-Based Surface Modification of Mesoporous Silica Nanoparticles as pH-sensitive Drug Delivery Vehicles for Cancer Therapy. J. Colloid Interf. Sci. 2016, 463, 279-287. (38) Liu, J. J.; Luo, Z.; Zhang, J. X.; Luo, T. T.; Zhou, J.; Zhao, X. J.; Cai, K. Y. Hollow Mesoporous Silica Nanoparticles Facilitated Drug Delivery Via Cascade pH Stimuli in Tumor Microenvironment for Tumor Therapy. Biomaterials 2016, 83, 51-65. (39) Zhao, J.; Feng, S. S. Effects of PEG Tethering Chain Length of Vitamin E TPGS with a Herceptin-functionalized Nanoparticle Formulation for Targeted Delivery of Anticancer Drugs. Biomaterials 2014, 35, 3340-3347. (40) Zhang, Z. P.; Feng, S. S. The Drug Encapsulation Efficiency, in Vitro Drug Release, Cellular Uptake and Cytotoxicity of Paclitaxel-Loaded Poly(Lactide)-Tocopheryl Polyethylene Glycol Succinate Nanoparticles. Biomaterials 2006, 27, 4025-4033. (41) Huijun, Z.; Hongbo, C.; Xiaowei, Z.; Zhongyuan, W.; Xudong, Z.; Yanping, W.; Yongfeng, G.; Jinxie, Z.; Kewei, L.; Ranyi, L.; Lintao, C.; Lin, M.; Si-Shen, F. Co-Delivery of Chemotherapeutic Drugs with Vitamin E TPGS by Porous PLGA Nanoparticles for Enhanced Chemotherapy Against Multi-Drug Resistance. Biomaterials 2014, 35, 2391-2400. (42) Tao, W.; Zeng, X. W.; Wu, J.; Zhu, X.; Yu, X. H.; Zhang, X. D.; Zhang, J. X.; Liu, G.; Mei, L. Polydopamine-Based Surface Modification of Novel Nanoparticle-Aptamer Bioconjugates for in Vivo Breast Cancer Targeting and Enhanced Therapeutic Effects. Theranostics 2016, 6, 470-484. (43) Win, K. Y.; Feng, S. S. Effects of Particle Size and Surface Coating On Cellular Uptake of Polymeric Nanoparticles for Oral Delivery of Anticancer Drugs. Biomaterials 2005, 26, 2713-2722. (44) Zhu, X.; Shan, W.; Zhang, P. W.; Jin, Y.; Guan, S.; Fan, T. T.; Yang, Y.; Zhou, Z.; Huang, Y. Penetratin Derivative-Based Nanocomplexes for Enhanced Intestinal Insulin Delivery. Mol. Pharmaceut. 2014, 11, 317-328. (45) Freitas, L. B. D.; Bravo, I. J. G.; Macedo, W. A. D.; de Sousa, E. M. B. Mesoporous Silica Materials Functionalized with Folic Acid: Preparation, Characterization and Release Profile Study with Methotrexate. J. Sol-Gel Sci. Techn. 2016, 77, 186-204. (46) Pang, J. M.; Zhao, L. X.; Zhang, L. L.; Li, Z. H.; Luan, Y. X. Folate-Conjugated Hybrid SBA-15 Particles for Targeted Anticancer Drug Delivery. J. Colloid Interf. Sci. 2013, 395, 31-39. (47) Kim, K.; Yang, E.; Lee, M.; Chae, K.; Kim, C.; Kim, I. S. Polydopamine Coating Effects On Ultrafiltration Membrane to Enhance Power Density and Mitigate Biofouling of Ultrafiltration Microbial Fuel Cells (UF-MFCs). Water Res. 2014, 54, 62-68. (48) Verma, A.; Stellacci, F. Effect of Surface Properties On Nanoparticle-Cell Interactions. Small 2010, 6, 12-21. (49) Zheng, Q.; Lin, T.; Wu, H.; Guo, L.; Ye, P.; Hao, Y.; Guo, Q.; Jiang, J.; Fu, F.; Chen, G. Mussel-Inspired Polydopamine Coated Mesoporous Silica Nanoparticles as pH-sensitive Nanocarriers for Controlled Release. Int. J. Pharmaceut. 2014, 463, 22-26. (50) Hikosaka, R.; Nagata, F.; Tomita, M.; Kato, K. Optimization of Pore Structure and Particle Morphology of Mesoporous Silica for Antibody Adsorption for Use in Affinity Chromatography. Appl. Surf. Sci. 2016, 384, 27-35. (51) Zhang, M.; Zhang, X.; He, X.; Chen, L.; Zhang, Y. A Self-Assembled Polydopamine Film On the Surface of Magnetic Nanoparticles for Specific Capture of Protein. Nanoscale 2012, 4, 3141-3147. (52) Iqbal, Z.; Lai, E. P. C.; Avis, T. J. Antimicrobial Effect of Polydopamine Coating On Escherichia 26


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Coli. Journal of Materials Chemistry 2012, 22, 21608-21612. (53) Zeng, X.; Liu, G.; Tao, W.; Ma, Y.; Zhang, X.; He, F.; Pan, J.; Mei, L.; Pan, G. A Drug-Self-Gated Mesoporous Antitumor Nanoplatform Based on pH-Sensitive Dynamic Covalent Bond. Adv. Funct. Mater. 2017, 27. (54) Zhuang, J. M.; Gordon, M. R.; Ventura, J.; Li, L. Y.; Thayumanavan, S. Multi-Stimuli Responsive Macromolecules and their Assemblies. Chem. Soc. Rev. 2013, 42, 7421-7435. (55) Chen, L. L.; Li, L.; Zhang, L. Y.; Xing, S. X.; Wang, T. T.; Wang, Y. A.; Wang, C. G.; Su, Z. M. Designed Fabrication of Unique Eccentric Mesoporous Silica Nanocluster-Based Core-Shell Nanostructures for pH-Responsive Drug Delivery. ACS Appl. Mater. Interfaces 2013, 5, 7282-7290. (56) Muharnmad, F.; Guo, M. Y.; Qi, W. X.; Sun, F. X.; Wang, A. F.; Guo, Y. J.; Zhu, G. S. PH-Triggered Controlled Drug Release From Mesoporous Silica Nanoparticles Via Intracelluar Dissolution of ZnO Nanolids. J. Am. Chem. Soc. 2011, 133, 8778-8781.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. (A) Synthesis of PEG-FA-conjugated polydopamine film through Oxidative Polymerization and Michael Addition Reaction. (B) Schematic illustration of doxorubicin-loaded MSNs-DOX@PDA-PEG-FA.

28


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure

1.

TEM

image

of

(A)

MSNs,

(B)

MSNs-DOX@PDA,

(C)MSNs-DOX@PDA-PEG-FA and (D)MSNs-DOX@PDA-PEG-FA treated with PBS of pH 5.6 for 15 days. (E) Pore size distribution from BJH adsorption of MSNs, MSNs-DOX and MSNs-DOX@PDA.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. XPS spectra of MSNs, MSNs@PDA and MSNs@PDA-PEG-FA. (A) – (C) narrow scan for N1s peaks. (D) – (F) narrow scan for Si2p peaks. (G) – (I) Surveys of all tested peaks.

30


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. (A) FTIR spectra of MSNs, MSNs@PDA, and MSNs@PDA-PEG-FA. (B) Thermogravimetric

analysis

(TGA)

curves

of

MSNs,

MSNs@PDA-PEG-FA.

31


MSNs@PDA,

and


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Figure 4. In vitro drug release profile of MSNs-DOX, MSNs-DOX@PDA and MSNs-DOX@PDA-PEG-FA in media with different pH value: (A) pH 7.4; (B) pH 5.6; (C) pH 2.0.

32


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (A) CLSM images of Hela cells after incubation with free DOX, MSNs-DOX@PDA-PEG, MSNs-DOX@PDA-PEG-FA and MSNs-DOX@PDA-PEG –FA + free folic acid for (A) 0.5 h, and (B) 2 h. (C)Flow cytometric histogram profiles of Hela cells after 2 h-incubation. (D) Quantification of Hela cells after incubation for 2 h. 1, 2, 3, 4 and 5 represent control, free DOX, MSNs-DOX@PDA-PEG and MSNs-DOX@PDA-PEG-FA and MSNs-DOX@PDA-PEG-FA + free folic acid, respectively. (*p< 0.05).

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Viability of Hela cells cultured with DOX-loaded NPs in comparison with that of free DOX at the same DOX dose: (A) 24 h, (B) 48 h, and (C) 72 h. (D) Viability of Hela cells cultured with drug-free MSNs and drug-free MSNs @PDA-PEG-FA for 72 h. (*p< 0.05, **p< 0.01).

34


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Ex vivo fluorescence images of major organs and tumor after systemically administration at 3 h and 24 h.

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure

8.

Anti-tumor

efficacy

of

DOX,

MSNs-DOX@PDA-PEG

Page 36 of 40

and

MSNs-DOX@PDA-PEG-FA on the SCID nude mice bearing Hela xenograft. (A) Images of tumors in each group taken out from the sacrificed mice at the end point of research. (B) Tumor growth curve after intravenously injected with Saline, drug-free MSNs

@PDA-PEG-FA,

DOX,

MSNs-DOX@PDA-PEG

and

MSNs-DOX@PDA-PEG-FA (*p

pH-Sensitive Delivery Vehicle Based on Folic Acid-Conjugated

Recommend Documents