Polydopamine-based surface modification of mesoporous silica nanoparticles as pH-sensitive drug delivery vehicles for cancer therapy

Journal of Colloid and Interface Science 463 (2016) 279–287

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Polydopamine-based surface modification of mesoporous silica nanoparticles as pH-sensitive drug delivery vehicles for cancer therapy Danfeng Chang a,b,1, Yongfeng Gao b,1, Lijun Wang b,1, Gan Liu b, Yuhan Chen c, Teng Wang a,b, Wei Tao b, Lin Mei b, Laiqiang Huang b,⇑, Xiaowei Zeng a,b,⇑ a

Department of Chemistry, Tsinghua University, Beijing 100084, PR 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, PR China c Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


A pH-sensitive drug delivery system

of mesoporous silica nanoparticles was prepared.
MSNs were surface modified by polydopamine for controlled release of desipramine.
The nanocarriers have good cytotoxicity and ASM inhibit efficiency.
The novel drug delivery system is promising for cancer therapy.

a r t i c l e

i n f o

Article history: Received 13 September 2015 Revised 2 November 2015 Accepted 3 November 2015 Available online 3 November 2015 Keywords: Mesoporous silica nanoparticles pH-sensitive Desipramine Polydopamine Cancer nanotechnology

a b s t r a c t A novel pH-sensitive drug delivery system of mesoporous silica nanoparticles (MSNs) which were modified by polydopamine (PDA) for controlled release of cationic amphiphilic drug desipramine (DES) was prepared. MSNs–DES–PDA were characterized in terms of size, size distribution, surface morphology, BET surface area, mesoporous size and pore volume, drug loading content and in vitro drug release profile. MSNs–DES–PDA had high drug loading content and pH sensitivity. The DES release profiles of MSNs– DES and MSNs–DES–PDA were totally different, and the drug release of MSNs–DES–PDA accelerated with increasing acidity. MSNs–DES–PDA can be internalized into cells. In vitro experiments demonstrated that MSNs–DES–PDA had higher cytotoxicity and inhibitory effects on acid sphingomyelinase than those of free DES. This drug delivery system was beneficial for controlled release and cancer therapy. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction ⇑ Corresponding authors at: Department of Chemistry, Tsinghua University, Beijing 100084, PR China (X. Zeng). E-mail addresses: [email protected] (L. Huang), zeng.xiaowei@sz. tsinghua.edu.cn (X. Zeng). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jcis.2015.11.001 0021-9797/Ó 2015 Elsevier Inc. All rights reserved.

Cancer has become one of the most serious global health problems in recent years, and millions of people die of cancer every year [1]. Nowadays, cancer is mostly treated by conventional approaches like chemotherapy, surgical resection and radiotherapy. However, these methods are highly aggressive, non-specific,

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and often accompanied by significant side effects, because they also show conspicuous toxicity to normal cells and tissues [2,3]. Having lower toxicity as well as higher efficiency and stability than those of conventional dosage forms, nanoparticles allow sustained and controlled delivery of anticancer agents, and also can be used to deliver drugs by altering signal transduction or modulating the tumor microenvironment [4–6]. Nanoparticulate drug delivery systems have been used to targetedly deliver drugs, to control the release of drugs, and to improve bioavailability and stability [7–9]. As delivery systems for drugs, nanoparticles preferentially accumulate and remain in tumors, unlike free drugs or small molecules that rapidly undergo renal filtration. As to the enhanced permeability and retention (EPR) effect, the retention time of drugs packed in nanoparticles is ten times that of free drugs at the tumor site [10,11]. Till now, nanoparticles have been widely used in drug delivery for cancer therapy. Mesoporous silica-based nanomaterial MCM-41 was discovered in 1992 [12]. Mesoporous silica nanoparticles (MSNs) contain a complex ‘worm-like’ network of channels throughout the interior, so they have large surface areas and extraordinarily high drug loading capacity. MSNs remain stable over broad ranges of temperature and pH, and can be used to deliver large doses of drug in a controlled manner [13,14]. In addition, the size, surface chemistry, shape, and mesoporous or hollow structure of MSNs can be controlled. MSNs also have high in vitro and in vivo biocompatibility, and can eventually be excreted from the body. For cancer therapy, MSNs are obviously superior to other nanoparticulate drug delivery systems [15,16]. MSNs, as drug delivery systems, have been used for delivery of chemotherapeutic drugs, therapy genes or co-delivery [17–19]. To block drug molecules inside the pores of MSNs and to control drug release, some ‘‘gatekeepers” are required on the surface of MSNs [20–22]. Polydopamine (PDA) is a biomimetic polymer which can form on a wide range of materials including polymers, ceramics,

noble metals, and semiconductors through self-polymerization of dopamine in an aqueous solution [23,24]. PDA coating, which is a well-documented gatekeeper on the surface of MSNs, is highly sensitive to pH. With this coating, drug molecules are blocked in MSNs at neutral pH and released at lower pH [25,26]. Lysosomes, as dynamic acidic organelles that contain hydrolytic enzymes capable of degrading intracellular components, are involved in cell death pathways [27]. Lysosomes are excellent pharmacological targets for killing cancer cells. Cationic amphiphilic drugs (CADs), such as desipramine (DES), have been developed to treat depression, allergies and hypertension. CADs are also applicable to cancer therapy [28], long-term use of which is safe, especially when compared with existing chemotherapeutics. CADs display cancer-specific cytotoxicity in vitro and in vivo, and can surmount multidrug-resistant phenotype. CADs exhibit cytotoxic activity and reverse tumor multidrug resistance by inhibiting acid sphingomyelinase (ASM), which is essential for lysosomal stability and survival of cancer cells, as well as for multidrug-resistant phenotype [29]. Probably directly inhibiting ASM, CADs lead to a generally dysfunctional lysosomal lipid homeostasis that severely affects the physiology of this cellular compartment, increases lysosomal fragility and causes lysosome membrane permeabilization, triggering cell death via apoptosis and apoptosis-like pathways [30,31]. As we known, nanoparticles are mainly ingested by cancer cells through endocytosis, and degraded in lysosomes [13] in which ASM is also located. In this study, we designed a strategy for cancer treatment as Fig. 1A, using PDA-coated MSNs as pH-sensitive nanocarriers loading DES. Nanoparticles were targeted to tumor by the EPR effect, and DES was released quickly at low pH in lysosomes and delivered directly to the target ASM. The MSNs were characterized, and the antitumor effects of DES-loaded MSNs and free drug were evaluated in vitro. The DES-loaded MSNs displayed higher antitumor activity than that of free drug.

Fig. 1. (A) Schematic of MSNs–DES–PDA, (B) FESEM image of MSNs, (C) TEM image of MSNs, (D) DLS size distribution of MSNs–DES–PDA, (E) FESEM image of MSNs–DES– PDA, and (F) TEM image of MSNs–DES–PDA.

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2. Materials and methods 2.1. Materials Tetraethylorthosilicate (TEOS), hexadecyl trimethyl ammonium bromide (CTAB), hydrochloride dopamine and hydrochloride desipramine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Ammonium fluoride (NH4F) was purchased from Aladdin Industrial Co., Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX) was bought from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). Acetonitrile and methanol were purchased from EM Science (HPLC grade, Mallinckrodt Baker, USA). ASM antibody (rabbit source) was from Cell Signaling Technology (CST, USA). b-Actin antibody was bought from Abmart Inc. (Shanghai, China). All other chemicals of the highest quality were commercially available and used as received. Human cervical carcinoma cell line HeLa was purchased from American Type Culture Collection (ATCC, Rockville, MD). 2.2. Synthesis of MSNs CTAB (1.82 g, 5 mmol) and NH4F (3 g, 81 mmol) were dissolved in 500 mL of water and heated up to 80 °C. Under vigorous stirring, TEOS (9 mL, 8.41 g) was added dropwise to the mixture solution that was then kept at 80 °C for 6 h. The solid product was centrifuged (12,000 rpm, 10 min), washed with water and ethanol several times and dried at 40 °C under vacuum. To remove the surfactant template (CTAB), the product was dispersed in 400 mL of ethanol containing 8 mL of hydrochloric acid (37%) and refluxed at 80 °C for 24 h [32]. This procedure was repeated twice to make sure that the surfactant was completely removed. The obtained MSNs were centrifuged and washed with deionized water. 2.3. Drug loading and PDA coating For DES loading, 100 mg MSNs were added to water solution of hydrochloride desipramine (5 mL, 10 mg/mL) and stirred for 24 h. The solution was centrifuged and washed with water to move the remaining DES from the surface of MSNs. DES-loaded MSNs (MSNs–DES) were dried at 40 °C under vacuum. DOX-loaded MSNs (MSNs–DOX) were also prepared by the same procedure. In order to modify the surface of DES-loaded MSNs with PDA, 50 mg MSNs–DES were dispersed in 25 mL of Tris–HCl buffer (pH 8.5, 10 mM) and then 25 mg hydrochloride dopamine was added. The mixture was stirred for 6 h in dark at room temperature. In the end, PDA-coated MSNs–DES (MSNs–DES–PDA) were centrifuged and washed several times with water to remove the unpolymerized dopamine [25]. MSNs–DOX–DPA and blank samples (MSNs–DPA) were also prepared by the same procedure. 2.4. Characterization of the nanoparticles The MSNs size and zeta potential were measured by Malvern Mastersizer 2000 (Zetasizer Nano ZS90, Malvern Instruments Ltd., UK). Before measurement, nanoparticles were appropriately diluted. All measurements were performed at room temperature after equilibration for 10 min. The surface morphology of MSNs was observed by field emission scanning electron microscopy (FESEM, JEOL JSM-6301F, Japan). To prepare samples for FESEM, MSNs were diluted, dropped on silicon slices, dried and coated with platinum layer by JFC-1300 automatic fine platinum coater (JEOL, Tokyo, Japan) for 60 s. The nanoparticles were further observed by transmission electron microscopy (TEM, Tecnai G2 20, FEI Company, USA). The sample

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was dropped onto a copper grid coated with a carbon membrane, and the grid was allowed to dry before characterization. N2 adsorption/desorption isotherms were measured at 196 °C by an ASAP 2020 accelerated surface area and porosimetry system (Micromeritics, USA). The specific surface area was calculated by the BET method. The pore size and pore volume were calculated by the BJH method according to the adsorption data of isotherm. Thermal gravity analysis (TGA) was conducted with Netzsch STA 449 (Germany) in an O2 atmosphere by heating the sample to 800 °C at the rate of 20 °C/min.

2.5. Drug loading content of MSNs–DES–PDA During the preparation of MSNs–DES–PDA, all the washings and supernatants after loading were collected and combined. The remaining DES was analyzed by HPLC [33] (LC 1200, Agilent Technologies, USA). A reverse-phase C18 column (150  4.6 mm, 5 lm, Agilent Technologies, USA) was used. The mobile phase consisted of acetonitrile, methanol and water (including 0.1% acetic acid) at the ratio of 45:45:10. The flow rate of mobile phase was 1 mL/ min. The column effluent was detected using a UV detector at 254 nm. The content of the remaining DES was calculated according to a calibration curve. The loading content of MSNs–DES–PDA was calculated by the following equation:

LC ¼

Weight of initial desipramine  Weight of remaining desipramine Weight of the nanoparticles

2.6. Drug release experiments in vitro In vitro drug release from drug-loaded MSNs was detected as described previously [34]. Ten mg freeze-dried MSNs–DES–PDA were dispersed in 1 mL of PBS at different pH values (7.4, 6.0 and 5.0). Then the dispersion was transferred into a dialysis bag (MWCO = 3500), and the bag was submerged in 10 mL of respective PBS and stirred at 37 °C. At designated time intervals, the supernatant was transferred into a test tube for HPLC analysis. The pellet was resuspended in 10 mL of fresh PBS solution and put back into the shaker for subsequent measurement. The cumulative release of DES from MSNs–DES–PDA was plotted against time.

2.7. Cell culture and cellular uptake of MSNs–DOX–PDA HeLa cells were cultured in monolayer in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 lg/mL streptomycin) in a humidified 5% CO2 atmosphere at 37 °C. In this study, DOX was used as a model fluorescent molecule loaded in MSNs for the qualitative investigation on cellular uptake by cancer cells such as HeLa cells. The cells were incubated with free DOX and MSNs–DOX–PDA (equivalent DOX concentration of 5 lg/mL) at 37 °C for 1 or 3 h, washed with cold PBS three times, and then fixed by 4% paraformaldehyde for 20 min. Then the cells were washed with PBS, stained with 40 ,6-diamidino-2-phenylin dole (DAPI) for 30 min and washed three times with PBS. The cells were observed by confocal laser scanning microscopy (CLSM, Olympus Fluoview FV-1000, Japan) with imaging software. The images of the cells were determined with differential interference contrast channel, blue channel (DAPI) excited at 358 nm and red channel (DOX) excited at 543 nm [35,36].

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2.8. In vitro cytotoxicity of MSNs–DES–PDA

3. Results and discussion

HeLa cells were seeded in 96-well plates at the density of 8  103 viable cells per well in 100 lL of medium and cultured overnight. Then the cells were incubated with the MSNs–DES– PDA suspension, free DES at equivalent drug concentrations ranging from 1 to 35 lg/mL and placebo MSNs and MSNs–PDA of the same nanoparticle concentrations for 24 and 48 h. At designated time intervals, 10 lL of MTT (50 mg/mL) was added per well. After the cells were incubated for additional 4 h, the medium was aspirated off and PBS was added gently to wash them. Then formazan crystals were dissolved with DMSO. Absorbance was measured at 490 nm using a microplate reader. Untreated cells were taken as a negative control with 100% viability and the cells without addition of MTT were used as blank to calibrate the spectrophotometer to zero absorbance.

3.1. Synthesis and characterization of nanoparticles

2.9. In vitro ASM-inhibiting effects of MSNs–DES–PDA The ASM-inhibiting effects of MSNs–DES–PDA were evaluated by Western blot. HeLa cells were seeded in 6-well plates at the density of 3  105 viable cells per well in 2 mL of medium and cultured overnight. Then the cells were treated with different concentrations of MSNs–DES–PDA suspension (equivalent DES concentrations: 1, 5, 10 lg/mL) for 12 h or at the same concentration (equivalent DES concentration: 10 lg/mL) for different times (6, 12, and 24 h). At designated time intervals, the medium was aspirated off, and the cells were washed with cold PBS, then lysed with 2 SDS loading buffer and boiled for 10 min. Proteins were separated by SDS–PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dried milk, incubated with primary antibody and secondary antibody (goat anti-rabbit) respectively, and then exposed on an X-ray film.

MSNs were synthesized by dropping TEOS to a mixture of CTAB and NH4F, followed by reflux with ethanol and HCl to remove the surfactant CTAB. Particle size and surface properties of nanoparticles play important roles in drug release, cellular uptake, pharmacokinetics and biodistribution [37]. In order to access the morphology of MSNs, FESEM and TEM were carried out. Fig. 1B and C presents the FESEM image and TEM image of MSNs respectively. All MSNs have nearly spherical shapes and porous surfaces, with the average size of approximately 140 nm. The size of MSNs was, as measured using dynamic light scattering (DLS), about 130.32 ± 5.51 nm (Table 1), being similar to the results of TEM and FESEM. Therefore, the size and shape of MSNs remained stable in solution and the dry state [13]. The different outcomes from those of organic polymer nanoparticles can be attributed to the tendency of the particles to shrinkage and collapse in the dry state [38]. MSNs–DES–PDA were prepared by adsorbing DES and surface modification with PDA. The loading of DES was about 71 mg/g. MSNs–DES–PDA were also studied by FESEM, TEM and DLS. Fig. 1E shows that the surface of MSNs–DES–PDA is much rougher than that of MSNs. Furthermore, the TEM image (Fig. 1F) shows obvious PDA coating on the MSNs surface, with a clear layer on the periphery of the particles. Because of the PDA coating, the diameter of MSNs–DES–PDA was slightly larger than that of MSNs. The DLS result also proved that the diameter of MSNs–DES–PDA (about 185.83 ± 4.54 nm) was smaller than the cut-off size of tumor vasculature pores (6200 nm) [39], falling within the range for easy accumulation in tumor vasculature because of the EPR effect. As shown in Table 1, the zeta potentials of MSNs and MSNs–DES–PDA are about 21.30 ± 4.17 mV and

Table 1 Characterization parameters of MSNs, MSNs–DES and MSNs–DES–PDA.

a b c

Sample

NPs sizea (nm)

Zeta potential (mV)

BET surface area (m2/g)

Pore volumeb (cm3/g)

Pore sizec (nm)

MSNs MSNs–DES MSNs–DES–PDA

130.32 ± 5.51 132.65 ± 7.18 185.83 ± 4.54

21.30 ± 4.17 16.52 ± 3.85 25.40 ± 3.73

253.84 103.70 33.72

0.51 0.33 0.11

2.76 2.15 –

NPs size was measured by dynamic light scattering. BJH cumulative pore volume for pores between 1.7 and 300 nm in width. The most probable pore size.

Fig. 2. (A) N2 adsorption/desorption isotherms, and (B) Pore size distribution from BJH adsorption of MSNs, MSNs–DES and MSNs–DES–PDA.

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Fig. 3. Thermogravimetric analysis (TGA) curves of MSNs and MSNs–PDA.

25.40 ± 3.73 mV, respectively. As indicated by the negative charges, the nanoparticles were stable in vivo through electrostatic repulsion, which was critical for a drug delivery system.

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The N2 adsorption/desorption isotherms and pore size distribution from BJH adsorption of nanoparticles are shown in Fig. 2. The adsorption/desorption isotherms seemed to be of type II [40]. The specific surface area, pore volume and the most probable pore size of MSNs, MSNs–DES and MSNs–DES–PDA are presented in Table 1. Compared with MSNs and MSNs–DES, all the pore parameters of MSNs–DES–PDA significantly decreased. The BET surface area was 253.84 m2/g, the pore volume was 0.51 cm3/g and the most probable pore size was, as evaluated by the BJH method, about 2.76 nm. Moreover, the pore size distribution of MSNs was rather narrow. With loading of DES, the BET surface area and the most probable pore size decreased to 103.70 m2/g and 2.15 nm, respectively. The BET surface area of MSNs–DES–PDA was merely 33.72 m2/g and the pore volume was 0.11 cm3/g due to the coating of PDA onto the surface of DES-loaded MSNs. In a word, the structural parameters of MSNs, MSNs–DES and MSNs–DES–PDA suggested that DES occupied the pore space of MSNs and DESloaded MSNs were coated with PDA. The weight loss of PDAcoated nanoparticles MSNs–PDA was 25.64% when heated in an O2 atmosphere to 800 °C, while that of MSNs was only 14.21% in the same temperature range (Fig. 3). The results of TGA further

Fig. 4. (A) In vitro release profiles of MSNs–DES, (B) In vitro release profiles of MSNs–DES–PDA, (C) TEM image of MSNs–DES–PDA treated with pH 7.4 for 12 h, (D) TEM image of MSNs–DES–PDA treated with pH 5.0 for 12 h, and (E) TGA of MSNs–DES–PDA treated with solutions at different pHs.

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demonstrated MSNs were successfully modified with PDA, the content of which was 11.43% [41].

released into lysosomes, which enhances the therapeutic effect and benefits cancer treatment.

3.2. pH-sensitive drug release in vitro

3.3. Cellular uptake of PDA-coated MSNs

In order to verify the drug blocking potency and pH sensitivity of PDA coating, the in vitro drug release profiles of PDA-coated particles MSNs–DES–PDA and non-coated particles MSNs–DES at different pH values were plotted. Furthermore, the TEM images of MSNs–DES–PDA with different pH values (pH 5.0, pH 7.4) that had been incubated for 12 h are displayed. Fig. 4A and B exhibit totally different DES release profiles of MSNs–DES and MSNs– DES–PDA. Without PDA coated, DES was released quickly at all tested pH values, freely diffusing from the pores of MSNs to the solution. The drug release profiles of MSNs–DES–PDA differed evidently at different pH values. At neutral pH (7.4) which is similar to normal physiological conditions like those of interstitial fluid or blood, only 20% of drug was released in 24 h and only 35% in 6 days. However, in acidic conditions (pH 6.0, pH 5.0) that simulated those of endosome and lysosome, about 45% and 70% of drugs were released in 24 h, respectively. This could be attributed to the PDA coating was partially detached from the surface of MSNs in the acidic condition. Thus, the coating blocked the pores as ‘‘gatekeepers” for drug controlled release. Moreover, the release rate of DES increased with rising acidity, being similar to that reported by Zheng and co-workers [25]. The PDA layer of MSNs–DES–PDA, which was relatively intact after treatment with neutral PBS (pH 7.4, Fig. 4C), was partially peeled off from the surface after being treated with acidic media (pH 5.0, Fig. 4D). The TGA curves of MSNs–DES–PDA treated with solutions at different pH values are presented in Fig. 4E, further indicating the PDA coating was detached from the surface of MSNs in the acidic condition [42]. In short, PDA coating was sensitive to pH and can be used as a drug delivery system. In the neutral condition, the coating can avoid premature drug release from MSNs during circulation. Once the particles enter cells by endocytosis, the drug can be quickly

The function of drug-loaded nanoparticles depends on internalization and sustained retention of the nanoparticles by cancer cells. In order to verify PDA-coated MSNs could be endocytosed by cancer cells, MSNs loading a hydrophilic fluorescent drug DOX (MSNs– DOX–DPA) were prepared by the same procedure as that of MSNs– DES–PDA. Fig. 5 presents the CLSM images of HeLa cells after 1 h (Fig. 5A) and 3 h (Fig. 5B) of incubation with MSNs–DOX–PDA or free DOX suspension in DMEM at equivalent DOX concentration of 5 lg/mL. The cells were imaged with differential interference contrast channel, blue channel (DAPI) excited at 358 nm and red channel (DOX) excited at 543 nm. As shown in Fig. 5, MSN–DOX–PDA can be ingested by HeLa cells, and internalized into the cytoplasm, corresponding to the way of nanoparticles entering cells by endocytosis. DOX was released from MSN–DOX–PDA and entered cell nuclei, as free DOX was mainly located in nuclei after cell uptake (Fig. 5C) [43]. Hence, PDA-coated MSNs can be internalized into HeLa cells in which they can release the drug from pores. 3.4. In vitro cell viability of MSNs–DES–PDA HeLa cells were used to study the in vitro cytotoxicity of MSNs– DES–PDA. Fig. 6 shows the in vitro cell viability of the drug formulated in MSNs–DES–PDA and DES at equivalent concentrations of 1, 5, 10, 25 and 35 lg/mL, respectively. The percentage of viable cells was quantitatively assessed by the MTT method. MSNs or MSNs– PDA did not have significant cytotoxicity against HeLa cells in vitro (Fig. 6). As previously reported, extreme concentration of MSNs (about 25 mg/mL) at some sizes showed cytotoxicity [44], but the required maximum concentration of MSNs herein was 500 lg/mL at which nearly no cytotoxicity was observed. Similar

Fig. 5. (A) Confocal laser scanning microscopy (CLSM) images of HeLa cells incubation with MSNs–DOX–PDA for 1 h, (B) CLSM images of HeLa cells incubation with MSNs– DOX–PDA for 3 h, and (C) CLSM images of HeLa cells incubation with free DOX for 1 h. DOX was red and the cells were stained by DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Viability of HeLa cells cultured with MSNs–DES–PDA compared with that of desipramine at the same drug dose and that of MSNs and MSNs–PDA with the same NPs concentration: (A) 24 h and (B) 48 h.

to our results, PDA coating was, as reported, nontoxic in various cell models and in vivo studies, and even PDA aggregates (100% PDA) were not toxic at 0.1 mg/mL [45]. Besides, with increasing drug concentration and incubation time, MSNs–DES–PDA and DES exerted enhanced therapeutic effects on HeLa cells. Meanwhile, MSNs–DES–PDA were significantly more cytotoxic than free DES at the same equivalent concentration both at 24 h and 48 h. Therefore, MSNs–DES–PDA killed cancer cells more effectively in vitro than the free drug did. IC50, the drug concentration at which the growth of 50% cells was inhibited, was evaluated by curve fitting of the cell viability data [46,47]. Table 2 shows the IC50 values of HeLa cells treated

Table 2 IC50 values of desipramine formulation in the free desipramine and MSNs–DES–PDA on HeLa cells following 24 and 48 h treatment, respectively (n = 6). Incubation time (h)

24 48

IC50 (lg/mL) Desipramine

MSNs–DES–PDA

22.31 ± 1.12 8.59 ± 0.56

7.21 ± 0.36 1.96 ± 0.13

with free DES and MSNs–DES–PDA for 24 h and 48 h. IC50 values of MSNs–DES–PDA at 24 h and 48 h were 7.21 ± 0.36 and 1.96 ± 0.13 lg/mL respectively versus those of free DES (22.31 ± 1.12 and 8.59 ± 0.56 lg/mL respectively), suggesting that the formers were 3.09- and 4.38-fold effective. Thus, MSNs–DES– PDA had much higher cytotoxicity against HeLa cells due to sustained drug release. 3.5. ASM-inhibiting effects of MSNs–DES–PDA As an important enzyme in sphingolipid metabolism, ASM plays key roles in apoptosis, immunity, development, and cancer [48]. ASM is a key target of antidepressants in the hippocampus. Therapeutic concentrations of the antidepressants reduce ASM activity and ceramide concentrations in the hippocampus [49,50]. In this study, to examine the influence of MSNs–DES–PDA on cancer therapy, their inhibitory effects on ASM were evaluated by Western blot after incubation of HeLa cells with different concentrations of MSNs–DES–PDA and free DES suspension in DMEM for 12 h or at the same concentration for different times (6, 12, and 24 h). Fig. 7A displays the ASM contents after different concentration treatments. Both MSNs–DES–PDA and free DES managed to inhibit

Fig. 7. (A) ASM inhibit efficiency of MSNs–DES–PDA and free desipramine at the drug concentration 1, 5 and 10 lg/mL for 12 h. (B) ASM inhibit efficiency of MSNs–DES–PDA and free desipramine at the same drug concentration 10 lg/mL for 6, 12, and 24 h.

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ASM, but the ASM-inhibiting effects of MSNs–DES–PDA surpassed those of equivalent free DES at all tested concentrations. Given that high-concentration MSNs–PDA was still nontoxic (vide supra), they also ought not to inhibit ASM, though the concentration of MSNs– PDA (300 lg/mL) was much higher than the maximum concentration of MSNs–DES–PDA. Fig. 7B displays the ASM-inhibiting effects of MSNs–DES–PDA and free DES at the same concentration after different times of treatment. With extended time, the inhibitory effects of MSNs– DES–PDA and free DES were augmented. However, since MSNs– DES–PDA were more effective than DES alone at each tested time point, DES was released quickly in the cells. Taken together, MSNs–DES–PDA inhibited ASM more effectively than free DES did, thus being preferred for cancer treatment.

[11]

[12]

[13] [14]

[15] [16]

[17]

4. Conclusions A pH-sensitive drug delivery system of mesoporous silica nanoparticles modified with PDA was prepared successfully for controlled release of cationic amphiphilic drug DES. MSNs–DES– PDA had the size of about 180 nm and high drug loading content. The in vitro drug release suggested that MSNs–DES–PDA were highly sensitive to pH, with the release rate increasing as the acidity rose. MSNs–DES–PDA were internalized into cells in which DES was released from the pores. MSNs–DES–PDA had higher cytotoxicity than that of free DES and MSNs–PDA may be biocompatible and nontoxic in vitro. In summary, the novel DES-loaded, PDAcoated MSNs are promising nanocarriers for cancer treatment. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 31270019 and 51203085), Guangdong Natural Science Funds for Distinguished Young Scholar (No. 2014A030306036), Natural Science Foundation of Guangdong Province (No. 2015A030313848), Program for New Century Excellent Talents in University (NCET-11-0275), Science, Technology & Innovation Commission of Shenzhen Municipality (No. CYZZ 20130320110255352), and Scientific and Technological Innovation Bureau of Nanshan District (No. KC2014JSCX0023A).

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