Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery

MSC-06465; No of Pages 8 Materials Science and Engineering C xxx (2016) xxx–xxx

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery Thu Thao Nguyen Thi a,b, Tuong Vi Tran a,b, Ngoc Quyen Tran a,b, Cuu Khoa Nguyen b, Dai Hai Nguyen b,⁎ a b

Institute of Research and Development, Duy Tan University, Da Nang City 550000, Vietnam Institute of Applied Materials Science, Vietnam Academy of Science and Technology, Ho Chi Minh City 70000, Vietnam

a r t i c l e

i n f o

Article history: Received 1 February 2016 Received in revised form 28 March 2016 Accepted 24 April 2016 Available online xxxx Keywords: Porous nanosilica Heparin Polyethylene glycol Cyclodextrin Self-assembly Host-guest complex Disulfide bond Doxorubicin Redox sensitive

a b s t r a c t Porous nanosilica (PNS) has been attracting a great attention in fabrication carriers for drug delivery system (DDS). However, unmodified PNS-based carriers exhibited the initial burst release of loaded bioactive molecules, which may limit their potential clinical application. In this study, the surface of PNS was conjugated with adamantylamine (A) via disulfide bonds (PNS-SS-A) which was functionalized with cyclodextrin-heparin-polyethylene glycol (CD-HPEG) for redox triggered doxorubicin (DOX) delivery. The modified PNS was successfully formed with spherical shape and diameter around 50 nm determined by transmission electron microscopy (TEM). DOX was efficiently trapped in the PNS-SS-A@CD-HPEG and slowly released in phosphate buffered saline (PBS) without any initial burst effect. Importantly, the release of DOX was triggered due to the cleavage of the disulfide bonds in the presence of dithiothreitol (DTT). In addition, the MTT assay data showed that PNS-SSA@CD-HPEG was a biocompatible nanocarrier and reduced the toxicity of DOX. These results demonstrated that PNS-SS-A@CD-HPEG has great potential as a novel nanocarrier for anticancer drug in cancer therapy. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Porous nanosilica (PNS) is received as a promising and broadly applicable of drug delivery system (DDS) due to their unique properties including high area and large pore volume, high chemical and thermal stability as well as excellent biocompatibility and biodegradability [1, 2]. Moreover, the guest molecules are effectively entrapped and protected by silica matrix which is capable of preventing enzymatic degradation, induced by pH and temperature changes of the surrounding medium [3]. Despite PNS's adequate impact on the drug loading capacity of PNS, the loading bioactive molecules would burst release, and be poorly dispersible from the unmodified PNS, leading to the loss of drug that actually reach cancer cells. These disadvantages limit the possible uses of unmodified PNS as novel drug delivery carriers. In order to overcome these challenges, surface modification of PNS by polymer grafting for DDS is receiving much attention, both the interfacial features of the modified nanoparticles can be engineered and the mechanical and thermal properties of the polymers can be enhanced at the same time. However, this preparation can precisely alter the release rate of drug for controlled release system, instead of deciding when or where to release drug [4].

⁎ Corresponding author. E-mail address: [email protected] (D.H. Nguyen).

In recent years, stimuli-responsive PNS as programmable drug delivery systems have attracted rapidly growing interest and drug release from PNS can be triggered by using appropriate stimuli. Enormous efforts have been devoted to formulate advanced PNS that are sensitive to either external stimuli (light, magnetic fields, and ultrasound) or internal stimuli (pH, temperature, and redox potential). Among these different types of stimuli, the redox stimulus is one the most effective strategies because the concentration of reducing agent, such as glutathione (GSH) in abnormal cells (2–10 mM) is 100 to 1000-fold higher than that in normal healthy cells (1–2 μM). In order words, the difference in concentration of antioxidants could offer a significant opportunity for redox sensitive system to deliver chemotherapeutic agents at the targeted tumor sites. For instance, Liang et al. reported the association of N-deacetylated heparin (HP) with PNS via disulfide bonds which would facilitate for the release of drug based on intracellular GSHtriggered [5–7]. By coating with heparin agents, PNS was proved to be able to elicit various promising characteristics such as to prevent or at least delay the process of phagocytosis, to stop the formation of blood clots as a part of inflammation as well as to inhibit angiogenesis and metastasis [8]. Additionally, this also has been shown to enhance the in vitro apoptosis and in vivo suppression of tumor growth and expansion considering for the efficient DOX delivery. Therefore, the redoxresponsive DOX-loaded PNS utilizing heparin (HP) as end-capping agent could possibly be functioned as a promised candidate for cancer target delivery system [9]. In addition, Zhao et al. developed a redox-

http://dx.doi.org/10.1016/j.msec.2016.04.085 0928-4931/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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responsive delivery based on 6-mercaptopurin (6-MP)-conjugated PNS via disulfide bonds and then also be modified with mPEG to elongate the blood circulation time and expand the dispersibility of nanoparticles [10,11]. Moreover, PEGylated PNS also can reduce the degree of hemolysis and protein adsorption and limit recognized immunology ability. The cumulative release of these drug delivery system reached N70% within 2 h in the presence of 3 mM GSH. The study introduced a new strategy to conjugate bioactive molecules and PEG to silica nanoparticles through disulfide bond to obtain redox-responsive drug release [12]. Besides, Lee and co-workers created a redox responsive system based PNS nanoparticles by using cyclodextrin (CD) as a gatekeeper with GSH via disulfide bridges (SS bonds). The conjugation of CD with PNS is not only to use CD as an impactful stimulus-responsive gatekeeper but also to improve the drug loading capacity. The results demonstrated that the adequate intracellular release of DOX by PNS-SSDOX-CD-PEG in the human lung cell line A549 which was found at significantly high levels of GSH. Furthermore, Quan Zhang et al. introduced a multifunctional mesoporous silica nanoparticles (MSNPs), the surface-immobilization of MSNP-SS-CD was achieved with a mixture of methoxyl poly(ethylene glycol) polymer (mPEG) functionalized with adamantine unit (mPEG-Ad) and PEG polymer functionalized with both folate unit (FA) and Ad unit (FA-PEG-Ad) based on the possibility of fitting with cyclodextrin cavity of Ad (Ad/β-CD), which creates a stable inclusion complex. These experiments indicated the potential properties of redox responsive PNS by surface PNS modification for targeted and controlled drug delivery in the treatment of cancer. More importantly, the unique structures PNS based stimuli-responsive delivery system would enable them to load large amounts of drug into mesoporous silica and then the pore entrances of PNS could be capped with different functional groups. In contrast, if the pore entrances of PNS system have been firstly blocked by caps before drug loading, encapsulation efficiency and drug loading capacity would be decreased. As a result, the blocking of the pore entrances by functional groups plays critical role in controlling drug loading [13,14]. Herein, we report a novel PNS based redox-responsive nanocarrier for controlling drug release in cancer treatment. The surface modification of PNS were prepared with adamantylamine (A) via disulfide bonds (PNS-SS-A) and then functionalized with cyclodextrin-heparinpolyethylene glycol (CD-HPEG) through a strong A–CD complexation for the formation of DOX delivery system (Fig. 1). The morphological and structural characteristics of the obtained samples were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), proton nuclear magnetic resonance (1H NMR), and Fourier transform infrared spectroscopy (FTIR). In a particular way, dithiothreitol (DTT) were carried out to reduce disulfide bonds in vitro release analysis. Methylthiazol tetrazolium (MTT) assay were used to determine the ability of the modified nanoparticles to minimize the toxicity

to HeLa cells of DOX. This study is expected to create significant opportunity for redox-responsive drug delivery systems in cancer therapy. 2. Experimental section 2.1. Materials Tetraethyl orthosilicate (TEOS, 98%), (3-aminopropyl) trimethoxysilane (APS, 97%), p-toluenesulfonyl chloride (TsCl), 1adamantylamine (A), doxorubicin (DOX, 99%), poly(ethylene glycol) methyl ether (mPEG, Mw 5000), dithiothreitol (DTT), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC, 97%), N,N-dimethyl formamide (DMF), ethylenediamine (EDA, 99%) were purchased from Sigma–Aldrich (USA). Cetyltrimethylammonium bromide (CTAB, 99%) was purchased from Merck (USA). 3,3′-Dithiodipropionic acid (DTDP, 99%), 3-aminopropythiethoxysilane (APS, 99%), heparin sodium (H), 4-nitrophenyl chloroformate (PNC, 97%) were purchased from Acros Organics (Belgium). β-Cyclodextrin (β-CD) was purchased from TCI (Japan). They were all used without further purification. 2.2. Preparation of PNS-SS-A Based on the literature with minor modification, the overall process of PNS-SS-A synthesis can be described in four steps (Fig. 2a): (1) PNS was synthesized by the sol-gel process, in which TEOS as silicon source, CTAB as structure-directing agents, ethanol as a solvent, water as a reactant, and ammonia (NH3) as catalyzed hydrolysis and condensation of TEOS. Briefly, deionized water (deH2O, 64 mL), ethanol (11.25 mL, 0.2 mol), CTAB (2.6 g, 7.1 mmol), and 2.8% NH3 solution (0.55 mL, 0.9 mmol) were mixed at 60 °C with a stir-bar for 30 min. TEOS (8 mL, 35.8 mmol) were added drop-wise to the surfactant solution within 5 min under stirring and the stirring was continued for another 2 h, and then filtered. The filtrate was dialyzed using a dialysis membrane (MWCO 6–8 kDa, Spectrum Laboratories, Inc., USA) against deH2O for 4 days at room temperature. The deH2O was changed 5–6 times a day and the resulting solution was then lyophilized to obtain PNS; (2) the amino-functionalized PNS (PNS-NH2) were prepared by stirring APS (1 mL, 5.7 mmol) and PNS (1 g) in toluene (30 mL) at room temperature under nitrogen environment for 24 h. The suspension was dialyzed using a dialysis membrane (MWCO 6–8 kDa) for 4 days against 2 M of acetic acid: ethanol (EtOH) (1:1 v/v, 250 mL). Acetic acid: ethanol solution was changed 5–6 times per day, and then the tube containing PNS was immersed into deH2O to remove acetic acid/EtOH for 1 day. The deH2O was changed 5–6 times a day and the resulting solution was lyophilized to generate PNS-NH2 as white powder; (3) the obtained PNSNH2 (1 g) and EDC (0.14 mL, 0.77 mmol) were dissolved in deH2O (20 mL) under stirring for 10 min. Then, DTDP (0.16 g, 0.77 mmol) in

Fig. 1. Schematic illustration the formation and redox-sensitive intracellular delivery of DOX-loaded PNS-SS-A@CD-HPEG nanoparticles.

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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Fig. 2. Synthetic route of the PNS-SS-A (a), CD-HPEG (b).

DMF (20 mL) were added into the mixture and the reaction was maintained for 24 h. After that, the sample was purified by a dialysis membrane (MWCO 6–8 kDa) against deH2O at room temperature for 4 days. The deH2O was changed 5–6 times a day and the solution was then lyophilized to obtain PNS-SS-COOH; (4) PNS-SS-COOH (1 g) and A (0.12 g, 0.77 mmol) in deH2O (40 mL) were mixed under the stirring condition, followed by the addition of EDC (0.11 mL, 0.64 mmol). The reaction was stirred at room temperature for 24 h, and then filtered. The sample was then dialyzed at room temperature for 4 days. The deH2O was changed 5–6 times per day and the solution was lyophilized to obtain PNS-SS-A [9,15–17].

dialysis membrane (MWCO 6–8 kDa) against deH2O at room temperature for 4 days. The deH2O was changed 5–6 times per day and the solution was then lyophilized for obtaining CD-H; (5) both mPEG-NH2 and CD-H were used with the support of EDC chemistry to obtain CDHPEG. CD-H (0.2 g, 7.4 μmol) and EDC (16 μL, 0.09 mmol) were dissolved in deH2O (10 mL) under stirring condition and mPEG-NH2 (0.54 g, 0.1 mmol) was then added into the mixture. After 24 h, the solution was filtered and dialyzed (MWCO 6–8 kDa) for 4 days. The deH2O was changed 5–6 times per day and the solution was lyophilized to collect CD-HPEG for future synthesis [18–21]. 2.4. Preparation of DOX/PNS-SS-A@CD-HPEG

2.3. Preparation of CD-HPEG The synthesis of CD-HPEG was carried out in five steps (Fig. 2b): (1) mPEG-NH2 was fabricated by using PNC chemistry under controlled conditions of temperature and vacuum environment. Briefly, mPEG (0.8 g, 0.16 mmol) was melted down at 65 °C under vacuum and PNC (0.04 g, 0.19 mmol) was added to the mPEG solution under constant stirring for 6 h. Thereafter, THF (20 mL) was used to dissolve excess mPEG at 40 °C and deH2O (10 mL) was added into the mixture to hydrolyze unreacted PNC. The obtained mPEG-PNC was slowly dropped into EDA solution (0.02 mL, 0.23 mmol) and the mixture was stirred at room temperature for 24 h. The solution was then dialyzed using dialysis membrane (MWCO 3.5 kDa, Spectrum Laboratories, Inc., USA) and lyophilized to obtain mPEG-NH2; (2) β-CD (30 g, 26.4 mmol) and 8.2 M of NaOH (10 mL) were dissolved in deH2O (200 mL) under constant stirring for 10 min. TsCl (5.04 g, 26.4 mmol) in acetonenitrile (15 mL) was added to the mixture. This reaction was maintained at 25 °C for 2 h and the pH of the solution was adjusted to about 8.0. The solid product was subsequently precipitated at 5–8 °C for 24 h, achieved under vacuum at room temperature, and dried at 70 °C for 2–3 days; (3) in order to synthesis of CD-NH2, CD-OTs (1.29 g, 1 mmol) was dispersed in DMF (30 mL) under stirring condition, followed by adding of EDA (2 mL, 30 mmol). The reaction was carried out at 75 °C under conditions of constant stirring and nitrogen-flushed for 24 h. After that, the mixture was evaporated and precipitated in acetone (500 mL) for several times. The solid product was dried under vacuum to obtain CDNH2; (4) CD-NH2 was conjugated into H by using EDC chemistry. Initially, H (0.2 g) and EDC (35 μL, 0.2 mmol) were dissolved in deH2O (10 mL) under stirring for 10 min. After that, the solution of CD-NH2 (0.26 g in 10 mL of deH2O) was added into the mixture and the reaction was maintained for 24 h. The solution was filtered and dialyzed by a

DOX has been loaded into the PNS-SS-A by sonication method. DOX (5 mg) and PNS-SS-A (20 mg) were dissolved in deH2O (1 mL) and sonicated for 10 min. Next, CD-HPEG (5 mg) was added in the mixture, sonicated for 10 min, and stirred for overnight. The sample was purified by dialysis membrane (MWCO 3.5 kDa) against deH2O for 3 h and then lyophilized. 2.5. Characterization FT − IR spectra were recorded on a Bruker Equinox 55 FTIR (Bruker Co., USA). XRD was performed using a Rigaku DMAX 2200PC (Rigaku Americas Co., USA) diffractometer equipped with Cu/Kα radiation at a

Fig. 3. FT-IR spectra of CD-HPEG (a), PNS (b), PNS-SS-A (c), and PNS-SS-A@CD-HPEG (d).

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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Fig. 4. 1H NMR spectra of CD-HPEG.

scanning rate of 4°/min (λ = 0.15405 nm, 40 kV, 40 mA). 1H NMR spectra were characterized by was performed on a Bruker Avance 500 (Bruker Co., USA). Morphology and size of particle were imaged by TEM using JEM-1400 (JEOL, Tokyo, Japan) at an accelerating voltage of 300 kV. The samples for TEM observations were prepared by placing a drop of solution in deH2O (1 mg/mL) onto a carbon-copper grid (300mesh, Ted Pella, Inc., USA) and air-dried for 10 min. Nitrogen adsorption–desorption isotherms were measured using a NOVA 1000e system (Quantachrome Instruments, USA). The samples were outgassed for 3 h at 150 °C before the measurements. The surface area was calculated by Barrett-Emmet-Taller (BET) model.

solution of DOX loaded PNS-SS-A@CD-HPEG in PBS (1 mL) containing 0.3 mg DOX were transferred to dialysis bags (MWCO 12-14 kDa), which were then immersed into the release medium (14 mL) in vials at 37 °C. The vials were placed in an orbital shaker bath, which was maintained at 37 °C and shaken horizontally at 100 rpm. At specific time intervals, 14 mL of the release medium was collected and an equal volume of fresh media was added. After lyophilization of the collected media, the released amounts of DOX were determined using UV– Vis spectrophotometer. To examine the ability of redox-agent to trigger the release of DOX, DOX release from PNS-SS-A@CD-HPEG into 5 mM DTT in PBS was carried out [22,23].

2.6. DOX loading contents and in vitro DOX release

2.7. MTT viability test

The drug loading efficiency (DLE) and drug loading content (DLC) were quantified using a UV–Vis spectrophotometer (NIR-V670, JASCO, Japan) and presented by Eqs. (1) and (2), respectively:

The MTT assay was carried out using mouse HeLa cells. HeLa cells were seeded in a 96-well plate at a density of 1 × 104 cells/well in 130 μL of Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin, and cultured 1 day at 37 °C. Then, the media was removed and the cells were incubated with samples. The cells were incubated for 48 h, followed by removing media, washing twice with PBS. MTT solution (25 μL) and culture medium (130 μL) were added into each well and the cells were cultured for 3 h. DMSO (130 μL) was added into each well to dissolve the precipitated. The cells cultured with media only were used as a control and assigned to 100% survival. The absorbance was measured at 570 nm using a multi-plate reader (SpectraMax M2e, Molecular Devices Co., USA). Cell viability of all other group was calculated by normalization its absorbance intensity to that of “Ctrl” group with the following

DLE ð%Þ ¼ weight of drug in particles=weight of drug feed initially  100 ð1Þ DLC ð%Þ ¼ weight of drug in particles=weight of particles and drug  100: ð2Þ The in vitro DOX release experiments were performed in PBS buffer (0.01 M, pH 7.4) at 37 °C using dialysis method. Firstly, suspension

Fig. 5. Transmission electron microscopy (TEM) micrographs of (a) PNS and (b) PNS-SS-A@CD-HPEG nanoparticles.

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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equation [23–26]:  
Cell viability ð%Þ ¼ ½Abssample −½Absblank = ½Abscontrol −½Absblank  100%:

ð3Þ

2.8. Statistical analysis The data were expressed as mean ± SD. The statistical evaluation of the data was performed by analysis of variance (ANOVA) followed by Student's t-test with p b 0.05 considered statistically significant. 3. Results and discussion 3.1. Characterization of PNS-SS-A@CD-HPEG The chemical structure of PNS-SS-A@CD-HPEG was determined by Fourier transform infrared spectroscopy. As shown in Fig. 3, the peaks around 1093 cm− 1 and 971 cm−1 were assigned to asymmetric stretching vibration of Si\\O\\Si bond and skeleton vibrations involving C\\O bond stretching of PNS, respectively. The peak intensity at 3425 cm−1 was assigned to the OH group on the surface of PNS. These absorption groups in PNS still existed after surface modification with polymers, indicating that either PNS-SS-A (Fig. 3c) or PNS-SS-A@CDHPEG (Fig. 3d) would keep the mesoporous structure of PNS after the process of modification. As seen in Fig. 3c, the characteristic absorption bands at 1723 cm−1 were attributed to the absorption of carboxyl groups. The sharp band at 2926 cm− 1 and the minor sharp band at 2855 cm−1 were assigned to CH2 antisymmetric and CH2 symmetric stretching of amantadine, respectively, suggesting the formation of PNS-SS-A. In addition, the weak signal at 562 cm− 1 was attributed S\\S stretching of DTDP. The peaks at 1640 cm−1 and 1572 cm− 1 were found to correspond to C_O stretching vibration of amide groups (amide I) and N\\H bending vibration band of amide groups (amide II), respectively. The appearance of amide I and amide II peaks indicated the existence of DTDP and ADA on the surface of PNS. In Fig. 3a, and d, the bands at 3413 cm−1 and 1174 cm−1 were assigned to O\\H stretching mode of C\\OH group and C\\O stretch in PEG and β-CD, respectively. The bands at 2887 cm−1 corresponded to \\CH2 stretching vibration in alkyl chains, and 1061 cm−1 were assigned to vibration of\\C\\O\\C groups of PEG. The intensity of the peaks at 1280 cm− 1 of SO2, 1147 cm− 1 of S_O stretching, 1445–1479 cm−1 of CH3 stretching

Fig. 6. Small-angle XRD patterns of PNS (a) and PNS-SS-A@CD-HPEG (b).

Fig. 7. Nitrogen adsorption–desorption isotherms for the as-synthesized PNS (a) and PNSSS-A@CD-HPEG (b).

band, 1370–1380 cm− 1 of CH2 stretching band of heparin were obtained. Moreover, the peaks at 1715 cm− 1 of C_O stretching of carboxylic group and 1634 cm− 1 of N\\H bending band (Amide II) were absorbed, resulting that heparin was prepared with CD and PEG. Overall results proved that CD-HPEG was conjugated to PNS-SS-A surface [24,27–29]. The CD-HPEG was characterized by 1H NMR analysis (Fig. 4). The peak at 4.7 ppm was assigned to the D2O used as a solvent. The peaks at 1.9 ppm (s, CH\\OH), 4.19 (s, NH\\SO3), 4.25 (s, CH\\COO), 3.1 (m, \\OCH3), 2.7 (m, CH2\\NH), 3.7–3.9 (m, C\\O), 3.6–3.7 (m, CH\\CH), 5.1 (s, C\\O\\C) were attributed to the H, CD-NH2 and mPEG-NH2, respectively. The presence of all the resonance signals demonstrated that CD-HPEG was successfully prepared [24,30–32].

Fig. 8. Release profiles of DOX from PNS (triangle) in PBS and from PNS-SS-A@CD-HPEG in PBS with (circle) and without (square) DTT (5 mM).

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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3.2. Particles properties of PNS-SS-A@CD-HPEG TEM of the unmodified PNS and the modified PNS are shown in Fig. 5a and Fig. 5b, respectively. The sizes of PNS and PNS-SS-A@CD-HPEG were approximately 40–45 nm and 45–50 nm, respectively. In biomedical applications, PNS should be utilized as a DDS for the purpose of delivering therapeutic agents to targeted cancer cells. Particularly, particle size plays a critical role in the pharmacokinetics of nanoparticles by affecting the clearance and particle bio-distribution. The efficiency of the cellular uptake decreases when increasing the particle size. It is stated that particles (diameter b 100 nm) are mostly removed by renal clearance, while particles (diameter N 200 nm) become accumulated in the spleen or taken up quickly by cells of the mononuclear phagocyte system (MPS) [33]. Besides, the particle size distribution within the size range of 10–100 nm is able to penetrate through extremely small

capillaries and to be non-selective uptake by MPS for prolonging circulation times, resulting in the high-efficiency targeting of drug. Moreover, the small size of PNS-SS-A@CD-HPEG would be responsible for the enhanced permeability and retention (EPR) effect results in higher particle concentration in tumor tissue. For instance, the particles range from 50 to 200 nm and N500 nm are easily taken up in a clathrinmediated endocytosis and raft-dependent pathway, respectively, whereas the small particles (40–50 nm in diameter) diffuse freely on the cell surface. Importantly, the surface area of PNS-SS-A@CD-HPEG has been increased after modification, which tends to effectively absorb therapeutic agents for targeted drug delivery. Therefore, PNS-SS-A@CDHPEG after hydrophilic surface modification with spherical form and the size of nearly 50 nm might serve as a nanocarrier with long-term circulation in the bloodstream and cellular uptake into cancer cells via endocytosis [34–36].

Fig. 9. (A) Images of HeLa cells incubated with PNS (a), PNS-SS-A@CD-HPEG (b) at different concentrations; free DOX (c), DOX loaded PNS (d), and DOX loaded PNS-SS-A@CD-HPEG (e) at different DOX doses observed under microscope for 48 h (scale bar = 80 μm) and (B) viability of HeLa cells incubated with (a) PNS (downward diagonal), PNS-SS-A@CD-HPPEG (dashed horizontal) at different concentrations and (b) free DOX (confetti), DOX loaded PNS (downward diagonal), and PNS-SS-A@CD-HPEG (dashed horizontal) at different DOX doses for 48 h. The cells were exposed to the samples for the indicated times. The data represent the mean values ± the standard deviation (SD) (n = 4).

Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085

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The ordered mesoporous structure of PNS was investigated by small angle XRD measurement and the results are shown in Fig. 6. Wellresolved diffraction peaks, assigned as (100) plane, could be clearly observed in the XRD patterns of PNS, which come from the typical peaks of MCM-41. The results indicated that the PNS had typical and ordered mesoporous structure like MCM-41. After the functionalization of PNS, there was a relatively weak diffraction peak (100), indicating the surface of PNS was fully covered by CD-HPEG [37]. The surface area and pore volume of PNS and PNS-SS-A@CD-HPEG nanoparticles were measured by N2 adsorption-desorption method (Fig. 7). There was a significant difference in nitrogen and structure parameters between PNS and PNS-SS-A@CD-HPEG, proving that CD-HPEG was exclusively grafted on the external surface of PNS. The prepared samples showed a typical IV feature, and PNS had a high specific surface area (SBET, and 561 m2/g) and pore volume (Vp, 0.24 cm3/g). After functionalization of CD-HPEG, however, the SBET and Vp of PNS-SS-A@ CD-HPEG were reduced to 130 m2/g and 0.12 cm3/g, respectively. 3.3. Loading and in vitro release of DOX Drug loading efficacy plays an important role in the formation of DDS and directly affects the therapeutic effect of the system [7,24,38]. The drug-loading amounts were determined by UV–vis spectroscopy and the results demonstrated that DLE and DLC of the PNS-SS-A@CDHPEG were 56.2 ± 2.5% and 10.5 ± 2.8%, respectively. In vitro release profile of DOX from PNS-SS-A@CD-HPEG was performed in order to evaluate release behavior of particle (Fig. 8). It is known that the concentration of GSH in intracellular cells is higher than extracellular cells [39]. In this study, the release rate of DOX from DOX/PNS-SS-A@CDHPEG nanoparticles was measured with the addition of DTT. Because DTT reduces the disulfide bonds of DOX/PNS-SS-A@CD-HPEG nanoparticles, it is expected that the presence of DTT increases the release rate. Fig. 8 shows the DOX release behavior of the DOX/PNS-SS-A@CD-HPEG nanoparticles with and without DTT over a period of 96 h. The release rate was obviously redox dependent and increased in the presence of DTT. After 96 h, about 78% of the DOX was released in the presence of DTT (5 mM), while b30% of the DOX was released in the absence of DTT. This means that the disulfide links was exceedingly sensitive to DTT, which may result in its enhanced efficacy in the intracellular delivery. These results demonstrated that the functionalization of CD-HPEG on PNS via disulfide bonds might undergo redox-sensitive dissociation and accelerate the DOX release rate in the intracellular region, but not in extracellular environments. 3.4. In vitro cytotoxicity Biocompatibility of a material is an important factor for its success in biomedical applications. It refers to the possibility of a biomaterial to perform its desired effect in respect of a biomedical therapy [40]. In this study, MTT assay was used to suggest biocompatibility of PNS-SSA@CD-HPEG nanoparticles in HeLa cells. The cell viability in cell culture medium can be observed under microscope using the 40 × objective (Fig. 9A-a, b). The results illustrated that both native and modified PNS showed no obvious cytotoxicity towards HeLa cells. Almost 100% of cells were still viable at 500 μg/mL of samples for 2 days, indicating that it is biocompatible (Fig. 9B-a). On the other hand, the cell growth was significant inhibited when the cells were treated with DOX loaded nanoparticles. A dose-dependent cytotoxicity was observed when individually incubated various doses of DOX, DOX loaded PNS, and DOX loaded PNS-SS-A@CD-HPEG with HeLa cells (Fig. 9A-c, d, and e). The majority of HeLa cells were killed when they were treated with DOX at concentration of 1 μg/mL for 2 days. The biocompatibility of PNS and toxicity of DOX are consistent with previous researches. It is expected that DOX encapsulated in PNS would be reduced its toxicity. As we could see in Fig. 9B-b, around 38% of cells were viable at an equivalent DOX concentration of 1 μg/mL. Free DOX was highly effective

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in vitro experiments due to its aqueous solubility and membrane permeability. It should be noted that, PNS and PNS-SS-A@CD-HPEG showed no toxicity against the cells. As a result, the PNS-SS-A@CD-HPEG could be safely used as a drug delivery vehicle for in vivo applications [41]. 4. Conclusion A novel surface modification of PNS with HPEG as a redoxresponsive controlled release system has been fabricated successfully via host-guest complex. The modified PNS nanoparticles were spherical in shape with a diameter range of 45 nm to 50 nm, which would be suitable for the development of drug delivery system. The resulting nanoparticles had drug loading efficiency and drug loading content of 56.2 ± 2.5% and 10.5 ± 2.8%, respectively. The release profile of the modified PNS nanoparticles showed sustained release of DOX in PBS and redox potential-sensitivity in the presence of DTT. More importantly, it was clearly that the nanoparticles could reduce the toxicity of DOX determined by MTT assay. Our results suggest that the modified PNS nanoparticles have potential as a redox-responsive DOX delivery system for cancer therapy. Acknowledgements This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.02-2014.83. References [1] M. Xie, H. Shi, Z. Li, H. Shen, K. Ma, B. Li, S. Shen, Y. Jin, A multifunctional mesoporous silica nanocomposite for targeted delivery, controlled release of doxorubicin and bioimaging, Colloids Surf. B: Biointerfaces 110 (2013) 138–147. [2] Y. Zhang, H.F. Chan, K.W. Leong, Advanced materials and processing for drug delivery: the past and the future, Adv. Drug Deliv. Rev. 65 (2013) 104–120. [3] J. Zhang, M. Niemelä, J. Westermarck, J.M. Rosenholm, Mesoporous silica nanoparticles with redox-responsive surface linkers for charge-reversible loading and release of short oligonucleotides, Dalton Trans. 43 (2014) 4115–4126. [4] I.I. Slowing, L.V.E. Juan, C.W. Wu, V.S. Lin, Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers, Adv. Drug Deliv. Rev. 60 (2008) 1278–1288. [5] C. Park, K. Oh, S.C. Lee, C. Kim, Controlled release of guest molecules from mesoporous silica particles based on a pH-responsive polypseudorotaxane motif, Angew. Chem. Int. Ed. 46 (2007) 1455–1457. [6] M. Zhao, A. Biswas, B. Hu, K.-I. Joo, P. Wang, Z. Gu, Y. Tang, Redox-responsive nanocapsules for intracellular protein delivery, Biomaterials 32 (2011) 5223–5230. [7] H. Li, J.Z. Zhang, Q. Tang, M. Du, J. Hu, D. Yang, Reduction-responsive drug delivery based on mesoporous silica nanoparticle core with crosslinked poly (acrylic acid) shell, Mater. Sci. Eng. C. Mater. Biol. Appl. 33 (2013) 3426–3431. [8] N.A.N. Tong, T.H. Nguyen, D.H. Nguyen, C.K. Nguyen, N.Q. Tran, Preparation of the cationic dendrimer-based hydrogels for controlled heparin release, J. Macromol. Sci. A 52 (2015) 830–837. [9] L. Dai, J. Li, B. Zhang, J. Liu, Z. Luo, K. Cai, Redox-responsive nanocarrier based on heparin end-capped mesoporous silica nanoparticles for targeted tumor therapy in vitro and in vivo, Langmuir 30 (2014) 7867–7877. [10] Z. Li, X.J. Loh, Water soluble polyhydroxyalkanoates: future materials for therapeutic applications, Chem. Soc. Rev. 44 (2015) 2865–2879. [11] D.H. Nguyen, J.S. Lee, J.H. Choi, Y. Lee, J.Y. Son, J.W. Bae, K. Lee, K.D. Park, Heparin nanogel-containing liposomes for intracellular RNase delivery, Macromol. Res. 23 (2015) 765–769. [12] Q. Zhao, C. Wang, Y. Liu, J. Wang, Y. Gao, X. Zhang, T. Jiang, S. Wang, PEGylated mesoporous silica as a redox-responsive drug delivery system for loading thiolcontaining drugs, Int. J. Pharm. 477 (2014) 613–622. [13] J. Lee, M. Kim, S.J. Jin, H. Lee, Y.K. Kwon, H.J. Park, C. Kim, Intracellular release of anticancer agents from a hollow silica nanocontainer with glutathione-responsive cyclodextrin gatekeepers, New J. Chem. 38 (2014) 4652–4655. [14] Y. Guo, W. Qin, Cyclodextrin-functionalized silica nanoparticles with dendrimer-like spacers for enantioselective capillary electrochromatography, Electrophoresis 35 (2014) 3549–3555. [15] A. Hakeem, R. Duan, F. Zahid, C. Dong, B. Wang, F. Hong, X. Ou, Y. Jia, X. Lou, F. Xia, Dual stimuli-responsive nano-vehicles for controlled drug delivery: mesoporous silica nanoparticles end-capped with natural chitosan, Chem. Commun. 50 (2014) 13268–13271. [16] I.I. Slowing, B.G. Trewyn, S. Giri, V.Y. Lin, Mesoporous silica nanoparticles for drug delivery and biosensing applications, Adv. Funct. Mater. 17 (2007) 1225–1236. [17] H.D. Nguyen, T.D. Nguyen, D.H. Nguyen, P.T. Nguyen, Magnetic properties of Cr doped Fe3O4porous nanoparticles prepared through a co-precipitation method using surfactant, Adv. Nat. Sci.: Nanosci. Nanotechnol. 5 (2014) 035017.

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Please cite this article as: T.T. Nguyen Thi, et al., Hierarchical self-assembly of heparin-PEG end-capped porous silica as a redox sensitive nanocarrier for doxorubicin delivery, Mater. Sci. Eng., C (2016), http://dx.doi.org/10.1016/j.msec.2016.04.085