Redox stimuli delivery vehicle based on transferrin-caped MSNPs for

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Redox stimuli delivery vehicle based on transferrincaped MSNPs for targeted drug delivery in cancer therapy Parthiban Venkatesan, Natesan Thirumalivasan, Hsiu-Ping Yu, Ping-Shan Lai, and Shu-Pao Wu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00036 • Publication Date (Web): 08 Mar 2019 Downloaded from http://pubs.acs.org on March 16, 2019

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ACS Applied Bio Materials

Redox stimuli delivery vehicle based on transferrin-caped MSNPs for targeted drug delivery in cancer therapy Parthiban Venkatesan,a Natesan Thirumalivasan,a Hsiu-Ping Yu,b Ping-Shan Lai*,b and Shu-Pao Wu*,a aDepartment

of Applied Chemistry, National Chiao Tung University, Hsinchu 300, Taiwan

bDepartment

of Chemistry, National Chung Hsing University, Taichung 402, Taiwan

*To whom correspondence should be addressed: Tel.: +886-3-5712121-ext 56506; Fax: +886-3-5723764; e-mail: [email protected] Tel.: +886-4-22840411 ext. 428 Fax: +886-4-22862547; e-mail: [email protected]

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Abstract Cancer has become one of the major diseases of human health around the world. Conventional antitumor drugs cannot specifically target cancers and result in serious side effects. In order to achieve a better therapy, innovative functional drug delivery platforms that will aid specific targeting for cancer cells need to be developed. In this study, transferrin (Tf), which can target cancer cells, is covalently anchored onto the surface of MSNPs via disulfide linkage, which is used for glutathione-triggered intracellular drug release in tumor cells. The successful functionalization of redox-responsive MSNPs is confirmed by using BET/BJH, TEM, TGA, NMR, and FT-IR. In addition, polyethylene glycol (PEG) is further grafted onto the surface of MSNPs to improve the biocompatibility and stability under physiological conditions for longer blood circulation. Our in vitro studies demonstrate that DOX-loaded MSNP–SS–Tf@PEG can selectively be internalized into cancer cells via Tf/Tf receptor interactions, and then DOX is released in HT-29 and MCF-7 cells triggered by high GSH concentration in tumor cells. Remarkably, in vivo studies demonstrate that DOX-loaded MSNP–SS–Tf@PEG can significantly inhibit tumor growth with minimized side effect through cell apoptosis determined by TUNEL assay, whereas MSNP–SS–Tf@PEG revealed no significant inhibition. In conclusion, DOX-MSNP–SS–Tf@PEG with active targeting moieties and redox-responsive strategy has been demonstrated as a great effective drug carrier for tumor therapy in vitro and in vivo.

Keywords: Mesoporous silica nanoparticles; transferrin; redox-responsive; in vivo doxorubicin; targeted drug delivery


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Introduction Great progress in new therapeutic methods for cancer treatment has been made recently.1,2 Because of more information about the physiological properties of chemotherapy drugs and their cellular uptake mechanisms, some adverse effects and reduced efficiency caused by common anticancer drugs remain as unsolved obstacles. Some deficits of cancer drugs include low water solubility, low stability, and unwanted side effects. To overcome the disadvantages, researchers are aiming at nanomedicine to offer a broad variety of tools and to increase the therapeutic efficiency of anticancer drugs. The improvement of therapeutic agents might provide more safe and effective treatments and reduce the toxicity of drugs to healthy organs. Over the past decades, several drug nanocarriers such as liposomes, nanoparticles,

4-14

and inorganic nanoparticles

15-18

3

polymeric

with diverse size, structure, and surface

characteristics have been developed. Mesoporous silica nanoparticles (MSNPs) have become an outstanding material for delivery systems because of unique features such as stability, high loading capacity, biocompatibility, and controlled drug-releasing ability

19-23

Specifically,

MCM41 nanomaterials have hexagonal arrangement of cylindrical mesoporous and are extensively studied. Since then, MSNPs have been applied as multifunctional delivery systems to carry therapeutic components to target cells and tissues upon contact with redox potential,

24-25

predetermined pH, 26-31 photoresponse, 32-34 and enzyme activities 35 as stimuli for cargo release. The delivery systems of theranostic compounds using responsive stimuli can also be applied for examining bio-spreading, targeting efficiency, and internalization pathways. Cancer has become a serious disease resulting in human death in the world. It is crucial to design and develop effective therapeutic strategies for cancer treatments. In this regard, understanding the physiological dissimilarity between health and tumor cells will allow us to develop effective stimuli-responsive drug delivery systems (SRDDS) for specific cancer therapy 36-40.

An effective drug delivery system should be designed in such a way to possess longer

circulation period, selective targeting ability and zero drug release in normal tissue. After nanomaterials were accumulated in tumor tissues by passive and active targeting or taken by

receptor mediated endocytosis by cancer cells, drug release from the nanomaterials should be rapid in response to the local environment. In particular, cancer cells have more transferrin (Tf) 3 ACS Paragon Plus Environment


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receptors than do normal cells because of an extraordinary metabolic request for fast growth. Transferrin (Tf), iron-binding blood plasma protein, has been used in targeting drug delivery systems. Herein, MNSP-Tf based nanocarrier was prepared via disulfide linkage, where Tf has dual functions: capping and targeting agents. Tf is biocompatible, biodegradable, nonimmunogenic and nontoxic. Hence, the drug delivery system based on Tf/MSNPs assures its potential for upcoming therapeutic applications. It is known that the level of glutathione (GSH) found in the intracellular matrix is two to three orders of magnitude higher than in the extracellular environments and has a critical role in cancer metabolism. In addition, Tffunctionalized nanoparticles can effectively aim at cancer cells through Tf receptors, while endogenous GSH can be an excellent stimulus to trigger drug release. 41-43

O H 2N

HN

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Figure 1. (A) Schematic illustration of surface functionalization on MSNPs. Stage I: thiol functionalization of MSNP-SH; stage II: further functionalization of MSNP-SS-NH2, stage III: DOX loading in MSNP-SS-NH2; stage IV: transferrin functionalization on DOX-MSNP–SS– NH2 V: PEG attachment on DOX-MSNP-SS-Tf. (B) Tf targeted recognition by Tf receptors on the cancer cells. Then receptor mediated endocytosis of nanoparticles into the cancer cells and glutathione-triggered drug release in the cancer cells. In this context, Tf-capped mesoporous silica nanoparticles (MSNPs) have been designed as a useful drug delivery system to target tumor cells with GSH-triggered drug release (Figure 1). Because of their uniqueness, MSNPs were used as an effective carrier with enhanced drug loading and reduced side effects in treatment. The mesoporous shells of MSNP were covalently linked with Tf via the disulfide bond to avoid the leakage of anticancer drugs during circulation. Tf is known as an effective tumor-targeting agent because of its Tf receptors, which are highly expressed on tumor cells, such as breast, pancreas, prostate, colon, and lung cells, but have lower expression in normal tissues.

44-45

Additionally, polyethylene glycol (PEG) is modified on the

surface of MSNPs to improve the stability and biocompatibility of MSNPs. Moreover, MSNPs could favorably go into the tumor cells through the recognition of Tf receptors. In addition, the concentration of GSH in the cellular matrix are two to three orders higher than that in extracellular surroundings. 46 After MSNPs entered the tumor cells, the gatekeeper Tf would be eliminated from the surface of MSNP by GSH-triggered disulfide breakage. We successfully 5 ACS Paragon Plus Environment


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demonstrated that doxorubicin (DOX)-loaded transferrin-capped MSNPs could precisely release DOX in MCF-7 (breast cancer) and HT-29 (colon cancer) cell lines. In addition, the cytotoxicity effect was studied, and the drug delivery system and bio-distribution profiles were analyzed and monitored by flow cytometry and confocal microscopy, respectively. Furthermore, we treated DOX-loaded MSNP–SS–Tf@PEG in an HT-29 tumor xenograft nude mouse model and demonstrated great anti-tumor efficacy. 2. Experimental Section 2.1 Materials 3-Mercaptopyltrimethoxysilane (MPTMS), thiopyridyldisulfide, 2-aminoethythiol hydrochloride, tetraethylorthosilicate (TEOS), 6-diamidino-2-phenylindole (DAPI), cetyltrimethylammonium bromide (CTAB) and 2-[methoxy(polyethyleneoxy)6-9propyl]trimethoxysilane (methoxy-PEG silane) were purchased from Alfa Aesar. Glutathione (GSH), N-hydroxysuccinimide (NHS), 3(ethyliminomethyleneamino)-N,

N-dimethylpropan-1-amine

(EDC),

2,-dithiodipyridine,

transferrin (Tf), dulbecco's modified eagle's medium (DMEM), doxorubicin (DOX), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and rhodamine B were purchased from Sigma-Aldrich. 2.2 Instruments NMR spectra data was obtained by on Varian Unity Inova 500 NMR spectrometer and Bruker DRX-300 NMR spectrometer. The X-ray diffraction pattern of MSNPs was collected by low angle powder X-ray diffraction system (Bruker D8 Discover XRD). The pore size distribution and surface area and of nanoparticles was measured by a Micromeritics ASAP 2020. UV-Vis spectra and Fluorescence spectra were obtained by an Agilent 8453 spectrophotometer and a HITACHI F-7000 fluorescence spectrophotometer, respectively. The TEM micrographs of nanoparticles were obtained by JEOL JEM-2010 Microscope. The zeta potentials of nanoparticles were measured by a Mavern Nanosizer. Infrared spectra of the samples were recorded by a Fourier transform infrared spectrometer (Bomem DA8.3 FT-IR). Fluorescence cell images were obtained by a confocallaser scanning microscope (TCS-SP5-X AOBS, Leica).


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2.3 Synthesis of MSNPs Synthesis of MSNP–SH In a regular procedure, CTAB (1 g) in 500 mL H2O was mixed with NaOH solution (3.5 mL, 2M). The mixture was stirred strongly at 80 ℃ and then TEOS (5 mL) was gradually added. After 2 hr stirring, the solution was centrifuged at 15000 rpm for 10 min and MSNPs were washed with deionized water/MeOH several times until the pH value of the solution was neutral. Silicon nanoparticles were desiccated at 80 °C under vacuum for 24 h. Further thiol modification on nanoparticles was carried out by the reaction of MSNPs (500 mg in 100 mL MeOH) with 3mercaptopyltrimethoxysilane (MPTMS, 0.5 mL) at 80 ℃ for 24 h under N2 atmosphere. The solution was centrifuged and silica nanoparticles were cleaned with MeOH and deionized water for 6 times. The surfactant CTAB in nanoparticles was removed by treating the MSNPs with HCl (3 mL)/MeOH (50 mL) for 24 h refluxing. The solution was centrifuged and silica nanoparticles were rinsed carefully with MeOH and deionized water. Silica nanoparticles MSNP-SH were dried at 80 °C under vacuum for 24 h. Synthesis of MSNP–SS–NH2 Further amino modified silica nanoparticles were achieved by the reaction of silica nanoparticles MSNP–SH with S-(2-aminoethylthio)-2-thiopyridine in 30 mL MeOH at room temperature for 24 h. The solution was centrifuged and silica nanoparticles MSNP– SS–NH2 were cleaned with deionized water/MeOH and desiccated at 80 °C under vacuum for 24 h. Synthesis of DOX-MSNP–SS–Tf@PEG MSNP-SS-NH2 (10 mg) was suspended in 10 mM phosphate buffer (pH 7.4). Rhodamine B (2 mg) or DOX (1 mg mL-1) was added to the solution and stirred in the dark for 48 h. DOX loaded MSNP-SS-NH2 was obtained by centrifugation and washed with deionized water for one time. Furthermore, transferrin (10 mg in 10 mM phosphate buffer, pH 7.4) was reacted with 3(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine

(EDC,

24

mg)

and

N-

hydroxysuccinimide (NHS, 14 mg) to activate the carboxylate groups at transferrin. The mixture was mildly stirred at 10 °C under nitrogen atmosphere in darkness for 2 h. Then, DOX loaded MSNP-SS-NH2 was added to the above solution with gentle stirring in the dark under nitrogen atmosphere at 10 °C for 24 h. The reaction mixture was added in a dialysis bag (20 kDa) against deionized water for 24 h to remove unreacted transferrin and small molecules. The nanoparticles 7 ACS Paragon Plus Environment


were lyophilized. Additionally, PEG modification on nanoparticles was carried out by the reaction of DOX-MSNP-SS-Tf (10 mg) with 30 µL of methoxy-PEG silane. The reaction was proceeded at 4 ℃ for 24 h and dialyzed for 24 h. The solution was centrifuged and silica nanoparticles were washed three times with deionized water. DOX-MSNP–SS–Tf@PEG were lyophilized and stored for further experiments and characterization tests. 2.4 Drug release test Drug release experiment of DOX loaded MSNP–SS–Tf@PEG was examined by dialysis method. DOX-MSNP–SS–Tf@PEG (5 mg) were suspended in 5 mL PBS (pH =7.4) with different concentration of GSH (0 mM, 10 mM and 20 mM) under ultrasonic. The suspended solution was added into a dialysis bag against 150 mL of PBS and stirred at 37 ℃. At certain time period, 2 mL of solution was taken to measure the absorbance at 480 nm by UV-vis spectrophotometer. 2.5 Cytotoxicity of MSNPs by MTT assay The cellular toxicity of Dox, MSNP–SS–Tf and DOX-MSNP–SS–Tf@PEG were tested by the MTT assay. The cell lines MCF-7, HEK 293 and HT-29 were cultured in a 96-well plate with DMEM medium at a density of 1 × 104 cells per well. After 24 h incubation, new medium (200 μL) containing free DOX, MSNP–SS–Tf, DOX-MSNP–SS–Tf@PEG in different amount (10 to 120 μg) were added to replace the old medium. After 24-hr incubation, the culture was added with MTT (20 μL, 0.5 mg mL−1). With 4-hr incubation, the culture medium was replaced by Sorenson's glycine buffer (0.1 M NaCl and 0.1 M glycine) and DMSO (200 μL). The absorbance at 570 nm in each well was measured. The cell viability was estimated by using the following equation: cell viability (%) = (mean of absorbance value of the treatment group) / (mean of absorbance value of the control group). 2.6 Fluorescence microscopic images MCF-7, HEK 293 and HT-29 cells were grown in 10 cm tissue culture dishes. DOX-MSNP–SS– Tf@PEG was added for 2 hr incubation. Then, the culture medium was taken away and the treated cells were washed with PBS three times. The fluorescent cell images were visualized by a confocal fluorescence microscope (Leica TCS-SP5-X AOBS). 2.7 Quantitative analysis by flow cytometry Flow cytometry was used to monitor the cellular uptake of nanoparticles. MCF-7 cells were grown in 6-well plates with the medium containg extra 10% fetal bovine serum (FBS) and 0.1 g 8 ACS Paragon Plus Environment

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L−1 kanamycin–streptomycin in 5 wt% CO2 at 37 °C. Then, the cells were treated with MSNP– SS–Tf@PEG and rhodamine B loaded MSNP–SS–Tf@PEG for 2 h. The treated cells were washed with PBS buffer and collected by centrifugation. The cells were suspended in PBS and monitored by a BD accuri c6 flow cytometer. 2.8 In vivo antitumor efficacy All experimental protocols and animal care were approved by Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing University, Taiwan, ROC (IACUC Permit No 105-088R). The antitumor growth inhibition experiment was carried out using Female BALB/cAnN.Cg-Foxn1nu/CrlNarl nude mice (5–6 weeks old, 20±2 g) provided from BioLASCO Taiwan Co., Ltd. (Taiwan). All mice were fed with standard food and filtered water and stayed in a temperature-controlled facility with a controlled light–dark cycle. HT-29 cells (1 ×107 cells/mL) were inoculated subcutaneously in the flank of the nude mice. Tumor volume was estimated as 1/2(4π/3) (L/2) (W/2)H, where H is the height, W is the width, and L is the length of the tumor. MSNPs were treated when the tumor volume was grown as 100 mm3 (day 0). All mice were divided into five groups (n=5 per group). The mice were injected with PBS (control group), free DOX (0.05 mg/mL), MSNP–SS–Tf@PEG or DOX-MSNP–SS–Tf@PEG (300 µL of 10mg/mL) via the lateral tail vein. Mice body weight and tumor size of mice were estimated for each mouse every 3 or 4 days. The percentage of tumor growth inhibition (TGI %) was estimated from the relative tumor volume at day 30. 2.9 Hematoxylin and eosin (H&E) and TUNEL staining Tumor tissues were cut and weighed after the mice were sacrificed. To evaluate the histological examination, tumor tissues were fixed with formalin (10%), embedded with paraffin and sliced into thin pieces. The segments were stained with hematoxylin and eosin (H&E). Cell apoptosis in tumor tissues was studied by a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay using a Click-iT® TUNEL Alexa Fluor Imaging Assay Kit (Thermo Fisher Scientific), and the experimental protocol was followed by manufacturer’s instructions. After the staining by Hoechst 33342 and Alexa Fluor® 488, cell fluorescence was observed by Leica SP5 confocal microscopy with an excitation wavelength at 405 and 488 nm, respectively. 2.10 Statistical analysis



All data were presented as mean ± standard deviation (SD). Statistical analysis was performed with OriginPro (version 7.5) via one-way ANOVA followed by Bonferroni’s tests. Statistics with a value of *p < 0.05 were considered significant. Result and Discussion MSNPs were synthesized by a sol–gel method in which tetraethyl orthosilicate (TEOS) is a precursor of silica and cetyltrimethylammonium bromide (CTAB) functions as a pore-generating agent (Figure 1). MSNPs were first functionalized with thiol group by surface grafting on MSNPs with 3-mercaptopyltrimethoxysilane to obtain MSNP-SH. Further reaction of MSNP-SH with S-(2-aminoethylthio)-2-thiopyridine hydrochloride formed a disulfide bond in MSNP-SSNH2. Tf-capped MSNPs (MSNP-SS-Tf) were achieved by the reaction of the carboxylic acid group in Tf and the amino group on the outer surface of MSNP-SS-NH2. Furthermore, methoxyPEG was also attached onto the surface of MSNPs to improve the stability and solubility of MSNPs. In addition, GSH functions as a reducing agent to effectively cleave the disulfide bond from the surface-functionalized nanoparticles and results in the release of cargo molecules. The characterization of MSNPs was done by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The collective results indicated that the average size of MSNP-SS-NH2 is 101 ± 10 nm (Figure S1 in the supporting information). The mesoporous structure found in MSNP-SS-NH2 nanoparticles is hexagonal symmetry observed by HR-TEM. Moreover, the interplanar distance between the d100 planes in the mesopore was calculated from powder X-ray diffraction and the TEM images (Figure 2 and Figure 3). According to the TEM images, the interplanar distance (d100) in MSNPSS-NH2 was found to be 3.95 nm, which is close to the value 4.08 nm calculated by Bragg’s law using 2θ = 2.16° for the d100 plane (Figure 3). After the capping with transferrin Tf, the mesoporous structure cannot be found in TEM images because of the shielding caused by the big size of transferrin (Figure S3 in the supporting information). Surface areas of MSNPs were studied by N2 adsorption–desorption isotherm. MSNP-SH and MSNP-SS-NH2 have a characteristic type-IV isotherm pattern with BET surface area of 989 m2 g−1, and 720 m2 g−1, respectively (Figure 4). Moreover, MSNP-SS-Tf loaded with rhodamine B had a type-II isotherm pattern with a small surface area, 389 m2g−1, because the mesopore in MSNP-SS-Tf was completely filled with rhodamine B. In addition, the pore diameter of MSNP-SS-NH2 10 ACS Paragon Plus Environment

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(2.04 nm) was smaller than MSNP-SH (2.14 nm) because of surface modification. The wall width of the mesopore calculated from the difference between the interplanar distance (4.08 nm) obtained from XRD, and the pore size (2.14 nm) obtained by the BJH method was found to be 1.94 nm. Furthermore, each modification on MSNPs was checked by the change of zeta potential (Figure S4). When MSNP-SH was modified to MSNP-SS-NH2, the zeta potential changed from −17.12 mV to +27.50 mV because of the modification of negative –SH groups by the positive – NH2 groups. Additional attachment of transferrin to MSNPs was confirmed from the zeta potential change +27.50 mV to −9.81 mV because of the isoelectric point value (pI = 5.6–5.8.) of Tf . 47

Figure 2. Particle images of MSNP-SS-NH2 observed by (A) TEM and (B) enlarged TEM. 10000

8000

X=2.161(d100)

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Figure 3. Powder X-ray diffraction pattern of MSPN-SS-NH2 and the lattice interplanar spacing of mesoporous structure.



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Figure 4. (A) The N2 adsorption–desorption isotherms and (B) pore distributions of MSNP-SH, MSNP-S-S-NH2 and MSNP-S-S-Tf. The functionalization on the surface of MSNPs can also be checked by FTIR, solid-state 13C

NMR, spectra, and TGA. In the IR spectra, the peak at 2568 cm−1 indicates the thiol group in

MSNP-SH, and the peak at 1562 cm−1 represents the NH2 bending frequency from MSNP-SSNH2. Further attachment of Tf on MSNPs increases the intensity of –NH peaks at 1562 cm−1 (Figure S5 in the supporting information). The 13C solid-state NMR spectrum of MSNP-SS-NH2 showed five carbon signals at 11.75, 23.40, 30.63, 40.24, and 51.69 ppm, indicating the attachment of 2-aminoethylthiol (Figure S6 in the supporting information). The TGA analysis of MSNP-SH, MSNP-SS-NH2, MSNP-S-S-Tf@PEG, and DOX-MSNP-S-S-Tf@PEG is shown in Figure 5. The weight losses of MSNP-SH and MSNP-S-S-Tf were 15.8% and 40.98%, respectively. Compared with MSNP-S-S-Tf, DOX-MSNP-SS-Tf had higher weight loss (47.28%), in which extra 6.30% weight loss comes from DOX loading. Furthermore, UV absorbance spectra of functionalized MSNP nanoparticles displayed peaks at 280 nm for Tf and 487 nm for DOX (Figure S7 in the supporting information). These results indicate the effective construction of Tf functionalization on MSNPs.


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100

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50

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o

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Figure 5. TGA analysis of (a) MSNP, (b) MSNP-SH, (c) MSNP-SS-NH2 (d) MSNP–SS– Tf@PEG and (e) DOX-MSNP-SS-Tf@PEG

80

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Figure 6. The drug release profile of DOX release from DOX- MSNP–SS–Tf@PEG after incubation in PBS solution. To check the drug loading capacity in MSNPs, nanoparticles MSNP-SS-NH2 were incubated with DOX or rhodamine B for 48 h at room temperature. Rhodamine B loaded in MSNP-SS-NH2 was estimated to be 10–13 µg per milligram of nanoparticles (Figure S8 and S9 in the supporting information). During the filling process, the best loading capacity ratio (rhodamine B to nanoparticles) was 2:10, in which less remaining rhodamine B was found; subsequently no noteworthy increase in loading amount was observed with increasing amount of rhodamine B. The redox-triggered drug release of MSNP-SS-Tf was monitored by UV–vis absorbance changes of DOX-loaded MSNP-SS-Tf@PEG. In Figure 6, there is no drug leakage 13 ACS Paragon Plus Environment


from DOX-loaded nanoparticles without addition of GSH, signifying that transferrin caps the mesopores and blocks DOX release. In the presence of 10 mM GSH while only 40% of DOX was released form DOX-MSNP-SS-Tf@PEG. When GSH was increased to 20 mM, 80% of DOX was released. This observation strongly supports that GSH can cleave the disulfide bond and results in the removal of Tf-capping, followed by the release of encapsulated drugs. Furthermore, the Tf gatekeeper could effectively prevent the leakage of DOX during the distribution of MSNPs. To examine the cytotoxicity of free-DOX, MSNP–SS–Tf@PEG, and DOX-MSNP-SSTf@PEG, the cell viability assay in MCF-7 and HT-29 and HEK-293 were used as illustrated. The breast cancer cell (MCF-7) and colorectal cancer (HT-29) cell lines that express higher amounts of Tf receptors on the cell surface were used as target cells. HEK-293, a human embryonic kidney cell line, has low Tf receptors in cells. In Figure 7, the MTT assay showed that MSNP-SS-Tf@PEG exhibited more than 80% cell survival at 120 µg mL−1 in each cell, indicating good biocompatibility of MSNP-SS-Tf@PEG. After DOX encapsulation, DOXMSNP–SS–Tf@PEG nanoparticles revealed a significant cell killing ability (>50%) compared with free-DOX-treated MCF-7 or HT-29 cells at high nanoparticle concentration of 120 µg mL−1 (Figure 7a and 7b). Interestingly, more than 80% survival rate was observed in DOX-MSNP– SS–Tf@PEG-nanoparticle-treated HEK293 cells (Figure 7c). The observation indicates that DOX-MSNP–SS–Tf@PEG nanoparticles caused less toxicity on HEK-293 because of less Tf receptors in cells. For MCF-7 or HT-29 cells, DOX-MSNP–SS–Tf@PEG is allowed to locate in the intracellular matrix because of higher Tf receptors. The Tf receptor plays an important role in internalization of Tf-conjugated nanoparticles. In addition, the high GSH level in cancer cells significantly increased DOX release from internalized Tf conjugated nanoparticles, thereby increasing cell selectivity and killing ability in MCF-7 or HT-29 cells.


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Figure 7. Cell viabilities of (A)MCF-7, (B)HT-29 and (C)HEK-293 cells treated with indicated nanoparticles and equivalent amount of free DOX for 24 h. The amount of free DOX used in MTT assay is the same with the amount of DOX loaded in MSNPs. Flow cytometry was used to measure the quantitative internalization of nanoparticles. MCF-7 cells were treated with MSNP-SS-Tf@PEG loaded with rhodamine B, free rhodamine B, and MSNP–SS–Tf@PEG for 4 h (Figure S10 in the supporting information). Comparatively, the fluorescence intensity of MSNP–SS–Tf@PEG loaded with rhodamine B was higher than MSNPSS-Tf@PEG. Rhodamine B loaded MSNP–SS–Tf@PEG displayed an enhanced rate of intracellular uptake (97.8%), higher than that of MSNP–SS–Tf@PEG (4.4%; Figure S10(b) and Figure S10(d)). The high fluorescence intensity from the MCF-7 cells indicated that the Tf receptor meditated endocytosis indeed. In order to understand how well MSNPs can be endocytosed, HEK-293, MCF-7, and HT29 cells were incubated with DOX-loaded MSNP–SS–Tf@PEG for 4 h at 37 C and monitored 15 ACS Paragon Plus Environment


by a confocal laser scanning microscope. Figure 8 shows the incubation of HEK-293, MCF-7, and HT-29 with DOX MSNP-SS-Tf@PEG. DOX is an anti-cancer drug and a fluorophore with red emission. In Figure 8B and 8C, red emission was found in nuclei of MCF-7 and HT-29. This observation indicates that MSNPs were allocated into the MCF-7 and HT-29 cells via receptor-mediated endocytosis. DOX-loaded MSNP–SS–Tf@PEG was designed to release DOX in cancer cells through GSH-induced disulfide bond cleavage because of relatively high concentration of GSH in several cancer cells. When MSNPs enter the cells through endocytosis, DOX can be released and transferred to nuclei. For HEK-293, no fluorescence was found in the cells but some tiny red spots were located outside of the cells (Figure 8A). This observation showed that DOX-MSNP–SS–Tf@PEG did not enter HEK-293 cells. HEK-293 cells are originally derived from human embryonic kidney cells, which are normal cells with less Tf receptor. High Tf receptor expression found in cancer cells such as MCF-7 and HT-29 cells can be used for tumor specific targeted diagnostic treatments.48 These collective data showed that DOXMSNP–SS–Tf@PEG can selectively target cancer cells, which have higher Tf receptors. We demonstrated that MSNP–SS–Tf@PEG is an effectual stimulus-receptive drug delivery system specifically for cancer cells that conquers the cytotoxicity problem of DOX in normal cells because of no specific target.


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Bright filed

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(A) HEK-293

(B) MCF-7

(C) HT-29

Figure 8. Fluorescence microscopic images of (A) HEK-293 (B) MCF-7 cells and (C) HT-29 cells treated with 50 µg/mLof DOX-loaded MSNP–SS–Tf@PEG nanoparticles for 4 hours at 37oC. The anti-tumor efficiency of DOX-MSNP–SS–Tf@PEG was explored by using nude mice bearing a subcutaneous HT-29 tumor model. The tumor-bearing mice were arbitrarily divided into four groups (n = 5) and treated with PBS saline, free-DOX, MSNP–SS–Tf@PEG, and DOX-MSNP–SS–Tf@PEG by intravenous injection. As shown in Figure 9A, tumors in the PBS control group increased rapidly, and there was no significant difference in volume with that in the MSNP–SS–Tf@PEG group, indicating no significant tumor inhibition using MSNP-SSTf@PEG. Mice treated with free DOX obviously exhibited tumor growth delay compared with mice in PBS saline or MSNP–SS–Tf@PEG groups, and the DOX-treated group could act as a therapeutic control in the HT-29 xenografted model. Interestingly, mice treated with DOXMSNP–SS–Tf@PEG showed significantly enhanced therapeutic efficacy compared with mice in the free DOX group. Figure 9B shows the relative body weight curves of mice in four groups, which show no obvious weight change during the experimental period. The tumor growth inhibitions (TGIs) of the MSNP–SS–Tf@PEG and free DOX group were 30.30% and 47.56%, respectively. It is noticed that the DOX-MSNP–SS–Tf@PEG group significantly increased the 17 ACS Paragon Plus Environment


TGI of DOX from 47.56% to 62.17%, which is consistent with the observation in tumor size in Figure 9C. Figure 9D shows the average tumor weight in four groups. On day 30, the average tumor weight in the DOX-MSNP–SS–Tf@PEG group was smallest compared with that in the PBS, free DOX, and MSNP–SS–Tf@PEG groups. It is reported that free DOX rapidly diffuses in most tissues after venous injection and then loses its bioactivity or is removed by the blood circulation and metabolism, resulting in cardiomyopathy and congestive heart problems and less tumor accumulation49. Remarkably, enhanced tumor growth inhibition in the DOX-MSNP–SS– Tf@PEG group was possibly due to the enhanced permeability and retention effect (EPR) of nanocarriers and selective targeting of the Tf-receptor-mediated endocytosis. In addition, PEGylation of MSNP nanoparticles could inhibit the drug release rate from mesoporous pores and increase blood circulating time and drug accumulation in the tumor site.50 Subsequently, the cell surface binds DOX-MSNP-SS-Tf@PEG, which might be internalized into the specific targeted cell via Tf-receptor-mediated endocytosis instead of passively diffusing through the cell membrane.51 Therefore, the DOX-MSNP–SS–Tf@PEG group selectively the targeted tumor site followed DOX release triggered by endogenous GSH. The resulting higher amount of DOX release into the nuclei successfully enhanced the antitumor efficacy. Thus, we demonstrated that the combination of active targeting based on Tf moiety and passive targeting of PEGylation MSNP revealed enhanced antitumor efficacy of DOX against colorectal cancer.


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Figure 9. Antitumor efficacy of DOX-MSNP–SS–Tf@PEG in vivo. (A) time-dependent relative tumor volume (n=5) *p < 0.05; (B) body weight changes; (C) tumor visual images after treatment of 30 days; (D) tumor weight inhibition effect; HT-29 cell-inoculated xenograft mice treated with PBS (control group), free-DOX, MSNP–SS–Tf@PEG or DOX-MSNP-SS-Tf@PEG. To further investigate the antitumor efficacy, hematoxylin and eosin (H&E) were used to stain tumor tissues treated with free DOX and DOX-MSNP–SS–Tf@PEG groups. As shown in Figure 10A, no noticeable change was observed in the tumor tissue between the PBS control and MSNP–SS–Tf@PEG group. The free DOX group exhibited considerable amount of apoptosis or necrosis. Moreover, DOX-MSNP–SS–Tf@PEG showed greater number of nuclei damage than that of free DOX group, indicating enhanced tumor apoptosis or necrosis. The therapeutic mechanism of apoptosis or necrosis was further examined using a terminal dexoynucleotidyl transferase (dUTP) nick end labeling (TUNEL) in tumor tissue, and results were shown in Figure 10B. Tumor tissues in both control and MSNP–SS–Tf@PEG groups exhibited less green 19 ACS Paragon Plus Environment


fluorescence signals, which indicated almost no cell apoptosis with regular nuclei. Remarkably, enhanced green fluorescence signals were observed in the DOX-MSNP–SS–Tf@PEG-treated group relative to that in the free DOX group. It is suggested that the active targeting ability of Tf moiety on the MSNP surface successfully delivered more DOX into tumor cells and resulted in increased cell apoptosis in tumor section. Thus, MSNP–SS–Tf@PEG can act as an excellent drug carrier for the combined strategies using active targeting drug delivery and redoxresponsive drug release for efficient antitumor therapy with less side effects.

Figure 10. (A) Histological examination for different treatments. Tumor tissues were stained with hematoxylin and eosin. (B) TUNEL detection of apoptotic cells in tumor tissues with different treatments. The tumor tissues were collected at 48 h after the last treatment. Nuclei were stained with Hoechst 33258 (red) and DNA fragments were labeled with fluorescein (green). Scale bar: 30 µm 20 ACS Paragon Plus Environment

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Conclusion In conclusion, we have constructed Tf functionalized MSNPs with biologically cleavable disulfide bonds for tumor-targeted drug delivery. Cancer cells have overexpressed a higher amount Tf-receptors located on the cell surface and have contained relatively high intracellular GSH. In this study, tumor-targeted drug delivery and GSH-controlled drug release are applied concurrently to achieve the best therapeutic efficiency with low side effect of the drugs. The mice studies have demonstrated that DOX-MSNP–SS–Tf@PEG can precisely transport DOX into tumor cells and successfully hinder the growth of tumor tissues, and then induce severe tumor cell apoptosis/death, as the results of combined effort of EPR effect and receptor-mediated endocytosis. Importantly, Tf-caped MSNPs show great biocompatibility in a wide range of concentration and increase targeting capability to tumor cells and cellular accumulation. Finally, PEG modified MSNPs can have long-term blood circulation in vivo and the enhanced permeability and retention (EPR) effect. To our best knowledge, Tf conjugated MSNPs via GSH triggered drug delivery system have demonstrated great biocompatibility for potential clinical applications against cancer. Acknowledgments The financial support was provided by the Ministry of Science and Technology (Taiwan) for supporting this research under the grant (MOST 107-2113-M-009-010) and (MOST 106-2113M-005-014-MY3), National Chiao Tung University and National Chung Hsing University for providing research facilities. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedure, SEM image and DLS particle size analysis of MSNP-SS-NH2, HRTEM images of MSNP-SS-NH2 and MSNP-SS-Tf, Zeta potential values of MSNPs, FT-IR spectra of MSNPs, MSNPs,

13C

Solid state NMR spectrum of MSNP-SS- NH2, UV-vis spectra of

the release profile of rhodamine B from rhodamine B loaded MSNP-SH,

cytometry histogram of MCF-7.


flow


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Graphic Abstract


Redox stimuli delivery vehicle based on transferrin-caped MSNPs for

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