Tunable Intracellular Degradable Periodic Mesoporous Organosilica

Dec 7, 2017 - Tunable Intracellular Degradable Periodic Mesoporous Organosilica Hybrid Nanoparticles for Doxorubicin Drug Delivery in Cancer Cells ...
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Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Tunable Intracellular Degradable Periodic Mesoporous Organosilica Hybrid Nanoparticles for Doxorubicin Drug Delivery in Cancer Cells Kummara Madhusudana Rao,*,† Surendran Parambadath,‡ Anuj Kumar,† Chang-Sik Ha,*,§ and Sung Soo Han*,† †

Department of Nano, Medical and Polymer Materials, School of Chemical Engineering, Yeungnam University, 280 Daehak-Ro, Gyeongsan, Gyeongbuk 38541, South Korea ‡ Department of Chemistry, TMJM Government College, Mahatma Gandhi University, Kottayam, Kerala, India § Department of Polymer Science and Engineering, Pusan National University, Busan 46241, South Korea S Supporting Information *

ABSTRACT: In this work, a dual (pH and redox)-sensitive cystamine-integrated periodic mesoporous organosilica (Cys-PMO) hybrid nanoparticle has been developed and subsequently loaded with doxorubicin (Dox) as an anticancer drug for intracellular cancer drug delivery. The formation of Cys-PMO was confirmed by FTIR, 13C (CPMAS), and 29Si MAS NMR spectroscopic techniques. X-ray diffraction and transmission electron microscopy confirmed that the Cys-PMO hybrid nanoparticles possessed mesoscopically ordered 2D hexagonal (P6mm) symmetry with cylindrical shape morphology. The N2 sorption isotherm showed that the Cys-PMO hybrid nanoparticles have a large surface area (691 m2 g−1), pore diameter (3.1 nm), and pore volume (0.59 cm3 g−1). As compared to conventional mesoporous silica materials and other PMO nanoparticles, the developed Cys-PMO hybrid nanoparticles have the capability of holding a high Dox content 50.6% (15.2 mg of Dox per 30 mg of Cys-PMO) at an optimized concentration (20 mg Dox) and avoid premature drug release under extracellular conditions. In vitro, the treatment of HeLa cells with Dox-encapsulated CysPMO hybrid nanoparticles results in a significantly greater cytotoxicity in response to intracellular acidic pH and a redox environment due to the degradation of disulfide bonds available in the framework of Cys-PMO hybrid nanoparticles. Further, confocal microscope images show the colocalization of Dox-loaded Cys-PMO hybrid nanoparticles inside the HeLa cells. Upon internalization inside HeLa Cells, the Cys-PMO use intracellular pH and redox environments to release Dox to the nucleus. Thus, the pH and reduction sensitivity of Cys-PMO hybrid nanoparticles make them suitable for intracellular drug delivery applications. KEYWORDS: intracellular degradable, nanoparticles, periodic mesoporous organo silica, cancer, drug delivery

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INTRODUCTION In cancer treatment, nanotherapeutics have received great attention due to systematic anticancer drugs that can be administered in high doses via sustained therapy. Recently, extensive research efforts have been made to develop several nanocarriers such as micelles, liposomes, and drug conjugates for cancer-targeted drug delivery.1 However, several biological barriers exist that hinder clinical translation of nanotherapeutics because of the burst release of the cancer drug and inability to reach the cancer target site.1 In order to avoid premature release at the extracellular level, recently, several researchers have developed multifunctional nanocarriers with intracellular degradable linkers such as micelles, nanogels, dendrimers, and mesoporous silica nanoparticles (MSNs) for drug delivery in cancer cells.2−8 Among various nanocarriers, multifunctional MSNs are superior to traditional nanocarriers because they can be designed to hold high amounts of drugs until interacting with specific functionalities, at which time drug molecules are released in a controlled manner.9 However, some of the carriers © XXXX American Chemical Society

could not hold a large amount of drug molecules. A series of multifunctional stimuli-responsive MSNs have already been reported. In these systems, the drug is triggered to be released from MSNs by external stimuli such as magnetic, electric, pH, photo, redox, or enzymatic conditions.10−13 However, the design of new bioresponsive nanocarriers has received great interest under particular pHs, and bioresponsive nanocarriers are well-suited for targeted drug delivery in cancer cells due to the acidic and redox environment of cancer cells in comparison with normal cells.14 Another exciting field of research in the expansion of MSNs is a class of new organic−inorganic nanocomposites known as periodic mesoporous organo silica (PMOs) hybrid nanoparticles.15,16 The incorporation of organic functionality into the framework structure of PMOs has allowed them to become Received: August 7, 2017 Accepted: December 6, 2017 Published: December 7, 2017 A

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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in a rotary evaporator. The product was confirmed by 1HNMR spectroscopy (Figure S2). Preparation of Cys-PMO Hybrid Nanoparticles. A solution of 7.2 mM of CTAB (2.62 g) and 14.4 mM NaOH (0.576 g) in double deionized (DI) water (108 mL) was prepared under with stirring conditions for 2 h at room temperature. A mixture of TEOS/Cys-bissilane was then added slowly to the reaction flask under stirring (Cysbis-silane was 8 mmol (20%) with respect to TEOS). Next, the reaction mixture was allowed to increase in temperature to 90 °C and held there for 24 h. The Cys-PMO hybrid nanoparticles were collected by filtration, washed with DI water and methanol, and dried at 60 °C. The CTAB extraction was carried out in ethanol (150 mL) using concentrated HCl (1 g) at 60 °C for 6 h. The final Cys-PMO product was collected by filtration, washed with DI water and methanol, and dried at 60 °C. Characterization. A Fourier transform infrared spectrum (FTIR; PerkinElmer, USA) was used for the existence of functional groups of the Cys-PMO hybrid nanoparticles. Small angle X-ray diffraction (XRD; AXN-Bruker) was performed using Cu Kα radiation in a 2θ range from 1.2 to 10°. The 29Si and 13C CP-MAS NMR spectra (Bruker-DSX 400) were recorded using a zirconia rotor spinning at 6 kHz (100.6 and 79.5 MHz resonance frequencies for 13C CP-MAS and 29 Si NMR, respectively). The 1H NMR (INOVA 400) spectra were recorded on a Varian NMR spectrometer at room temperature at a frequency of 400 MHz. The morphology of the Cys-PMO hybrid nanoparticles was characterized by scanning electron microscopy (SEM; JEOL-6400) at an acceleration voltage of 20 kV. Before analysis, the sample was coated with gold (deposition rate 30 s). Further, the size and morphology of Cys-PMO hybrid nanoparticles were analyzed using high-resolution transmission electron microscopy (HR-TEM; FEI-Tecnai F20) at an accelerating voltage of 200 kV. For this, a drop of Cys-PMO hybrid nanoparticles (dispersed in ethanol) was deposited on the surface of a copper grid and then dried under a lamp. The particle size of Cys-PMO hybrid nanoparticles was measured using dynamic light scattering on a Malvern Zetasizer instrument. The Brunauer−Emmet−Teller (BET) method was used for specific surface area and pore volume of Cys-PMO hybrid nanoparticles. The pore size distribution of Cys-PMO hybrid nanoparticles was calculated according to the Barrett−Joyner− Halenda (BJH) method. Thermogravimetric analysis (TGA; Q600SDT TA Instruments, 30−800 °C) was carried out for Cys-PMO hybrid nanoparticles under a N2 atmosphere (flow rate of 100 mL/ min) at a heating rate of 10 °C/min. Dox Loading and in Vitro Release Experiments. Typically, 50 mg of Cys-PMO hybrid nanoparticles were dispersed into a vial containing 5 mL of PBS with Dox (2, 5, 10, 15, 20, and 30 mg). The resulting solutions of Dox and Cys-PMO hybrid nanoparticles max 495 nm. The drug loading content (%DLC) and encapsulation efficiency (%EE) were calculated by the following eqs 1 and 2).

efficient drug delivery systems due to their biodegradability, good hemocompatibility, and allowance of the absorption and release of drugs in a controlled manner.17 At present, PMOs as drug delivery systems, based on various organic or inorganic functionalities of silane precursors, have been constructed. It was reported that functional biodegradable frameworks integrating PMOs have been used in imaging, multicargo drug delivery, tumor-targeted drug delivery, and enzymatic degradation-targeted drug delivery applications.18−20 Lu et al. developed thio-ether integrated PMOs that have been used in dual responsive drug release for targeted drug delivery in cancer cells.21 In another study, biodegradable PMOs were developed by the incorporation of disulfide linkers in the framework of PMOs for in vitro cancer therapy.17 Such carriers show improved drug delivery performance in the intracellular cancer cells under redox conditions. Therefore, PMOs have great potential in drug delivery applications. However, previously reported PMOs were not able to allow higher amounts of drug molecules because of a lack of multifunctional moieties in the framework of PMOs. In view of the importance of PMOs for drug delivery applications, herein, for the first time we attempted to increase the multifunctional moieties (pH and redox-sensitive) in the framework of PMOs as both pH- and redox-responsive drug delivery systems in order to improve both payload and targeted drug delivery performance in cancer cells without affecting the normal cells. Therefore, in this study, a cystamine-modified organo silane functional moiety is introduced in the framework of PMOs by a co-condensation method. The developed Cys-PMO hybrid nanoparticles were analyzed for the loading of doxorubicin (Dox) as a cancer drug for intracellular drug delivery performance in a human cervical carcinoma (HeLa) cells. The system has disulfide (−S−S−) bonds and more carboxylic functionalities. Therefore, CysPMO hybrid nanoparticles are beneficial for the high loading of Dox (via formation of the complexation of carboxylic groups of Cys-PMO and amino groups of Dox) as compared to other drug delivery carriers.18−21 In addition, the system exhibits both a pH- and redox-responsive nature. So, Cys-PMO hybrid nanoparticles could be more stable at extracellular conditions than in the intracellular cancer cell environment. We hypothesized that these Cys-PMO hybrid nanoparticles could be more effective carriers for the intracellular cancer delivery of Dox to HeLa cells to improve the anticancer activity with a minimized side effect.

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EXPERIMENTAL METHODS

%DLC =

Materials. Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), cystamine hydrochloride (Cys.HCL), 1,4dithiothrietol (DTT), glutathione reduced ethyl ester (GSH-OEt), anhydrous tetrahydrofuran, toluene, and penicillin G-streptomycin were purchased from Sigma-Aldrich. 3-(Triethoxysilyl)propylsuccinic anhydride (TEPA) was purchased from Alfa Aesar. Cystamine was prepared as reported elsewhere and confirmed by 1H NMR spectroscopy (Figure S1).22 Dulbecco’s Modified Eagle’s Medium (DMEM) with phenol-red and fetal bovine serum (FBS) were purchased from Gibco (Invitrogen Ltd., USA). Cell Culture. HeLa cells from American Type Culture Collection (ATCC) were cultured using DMEM medium with phenol-red containing 10% FBS and 1% penicillin G-streptomycin. Cells were maintained in a humidified incubator at 37 °C under a 5% CO2 atmosphere. Preparation of Cys-bis-silane Precursor. A round-bottom flask was charged with Cys (0.225 g, 1 mmol) and TEPA (0.608 g, 2 mmol) in 20 mL of THF. The resulting mixture was stirred for 3 h at room temperature. Then, the THF solvent was stripped off under a vacuum

%EE =

Amount of Dox in the Cys − PMO × 100 Amount of Cys − PMO

Amount of Dox in the Cys − PMO × 100 Amount of Dox used for formulation

(1)

(2)

The in vitro release of Dox from Cys-PMO hybrid nanoparticles was carried out at various conditions such as extracellular conditions (pH 7.4) and intracellular cancer environment (pH 5.5 and DTT 10 mM). To examine the Dox released from the Cys-PMO, 50 mg of Doxloaded Cys-PMO hybrid nanoparticles was placed in tubes with 3 mL of medium (PBS, pH 7.4, acetate buffer at pH 5.5, and both pH 5.5 and DTT 10 mM) and shaken using a shaker at 37 °C. After different intervals of time, the released medium was collected by centrifugation (1000 rpm at 5 °C for 5 min), and fresh medium was added for further releasing analysis. The collected supernatant medium was analyzed by UV−vis spectrophotometrically at a wavelength of λmax 495 nm. The release studies were carried out in triplicate to calculate the standard deviation. B

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Cytotoxicity Studies. Prustoblue cell viability assay was used for cytotoxicity of Dox and Dox-loaded Cys-PMO hybrid nanoparticles. HeLa cells were trypsinized and counted manually using a hemocytometer. Initially, the HeLa cells were seeded into 96-well plates at a density 10 000 cells/cm2, and the cells were allowed to attach using the appropriate cell culture medium (100 μL) for 24 h. The culture medium was removed, followed by the addition of various concentrations of Dox and Dox-loaded Cys-PMO solutions and incubated for 48 h. A total of 100 μL of Prustoblue reagent (1:10 dilution with DMEM medium) was added and then incubated for 1 h under dark conditions. The absorbance of each well was measured at wavelengths of 570 and 600 nm (Bio-T Instruments, Inc., USA). Cell viability was determined by normalizing the absorbance of each test well to the average intensity of control cells. All assays were performed in triplicate. The mean values and their standard deviation values are reported. Cell Uptake Studies. HeLa cells at a density 5000 cells/cm2 were seeded onto coverslips and incubated for 24 h. Then, 200 μL of 10 μM Dox and Dox-loaded Cys-PMO hybrid nanoparticles was added to each well and incubated for 6 h. Afterward, the media were removed, and then 500 μL of 4% formaldehyde solution was added to wells and incubated for 10 min. The wells were washed with PBS (pH 7.4). The coverslips were carefully removed from each well and mounted onto glass slides using Fluoroshield with DAPI (Sigma-Aldrich) in order to counterstain the nuclei with DAPI. The cell internalization and intracellular distribution were confirmed by the red fluorescence intensity of Dox and Dox-loaded Cys-PMO hybrid nanoparticles using confocal laser scanning microscopy images (CLSM; Model 700; Carl Zeiss, Oberkochen, Germany).

Scheme 1. Schematic Representation of Preparation of Dox Loaded Cys-PMO Hybrid Nanoparticles

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RESULTS AND DISCUSSION Preparation of Cys-PMO Hybrid Nanoparticles. In cancer drug delivery, stimuli-responsive nanocarriers are more important to transporting anticancer drugs to a particular target site due to the greater availability of acidic pH and abundant redox potential than those of normal cells.6 A few studies have reported on PMO hybrid nanoparticles for cancer drug delivery applications via pH-, enzyme-, and redox-activated intracellular drug release in cancer cells.17−21 In this work, we were interested in incorporating both redox- and pH-activated organic functional moieties in the framework of PMO hybrid nanoparticles that were subsequently loaded with Dox as an anticancer drug. For this, a new cystamine silane (Cys-bissilane) precursor was synthesized by reacting TEPA with Cys (Scheme S1), and the formation of a Cys-bis-silane precursor was confirmed by 1H NMR spectroscopy (Figure S2). In the next step, Cys-PMO hybrid nanoparticles were prepared by a co-condensation method using a Cys-bis-silane precursor as the source of the organosilane bridging part, TEOS as the parent silica source, and CTABr as the structural agent (Scheme 1). Due to the availability of carboxylic functional groups, a large amount of Dox was loaded into Cys-PMO because of complexation of the amino groups of Dox with the carboxylic groups of Cys-PMO. The release of Dox from Cys-PMO hybrid nanoparticles was due to the destabilization of the Cys-PMO network in response to pH and redox stimuli. Moreover, these Cys-PMO hybrid nanoparticles were highly stable in the extracellular environment (pH 7.4). Therefore, the developed tunable intracellularly degradable Cys-PMO hybrid nanoparticles are suitable for Dox drug delivery in cancer cells. Characterization of Cys-PMO Hybrid Nanoparticles. The formation of Cys-PMO hybrid nanoparticles was confirmed by FTIR, 13C CP-MAS NMR, and 29Si MAS spectroscopic techniques, as shown in Figure 1. In Figure 1a, Cys-PMO hybrid nanoparticles showed IR characteristic peaks at 1059 and 795 cm−1, corresponding to Si−O−Si and Si−O

group vibrations, respectively. The peak at 1605 cm−1 was attributed to an amide stretching vibration, and a peak at 1625 cm−1 can be assigned to the bending vibration of O−H of CysPMO. The characteristic peak of the CO group of a carboxylic group was found at 1732 cm−1. The existence of all functional groups of the Cys-bis-silane precursor in the CysPMO networks confirmed the formation of Cys-PMO. Moreover, the disulfide bond (S−S) stretching vibration did not appear in the IR spectra due to the sensitivity limit to detect the less intensive S−S bond. The solid-state 13C (CP-MAS) NMR spectrum (Figure 1b) of the Cys-PMO material confirmed the successful formation and incorporation of multifunctional Cys moieties in the framework of the CysPMO hybrid nanoparticles. The spectrum exhibited six signals. From this, three important signals (0−50 ppm) were assigned to Si−CH2−CH2−CH2− groups of TEPA (numbers 1, 2, and 3). The broad peak at 176 ppm represents the presence of two carbonyl carbon atoms, numbers 6 and 7. The chemical shift value at 54 ppm represents the tertiary carbon atom number 4 attached to the carboxylic acid carbon atom. The signal at 41 ppm was observed corresponding to the carbon atoms attached to the amide carbon and nitrogen atoms (numbers 5 and 8). The NMR signal of the carbon atom (number 9) directly attached to the disulfide group seems to be merged with the third carbon atom in the propyl group at 35.5 ppm. As from the 29 Si MAS NMR spectrum (Figure 1c), the strong resonances at C

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 2. N2 adsorption−desorption isotherm and pore size distribution curve of Cys-PMO hybrid nanoparticles.

Po = 0.30−0.93 and obvious H1 hysteresis loop characteristics of mesoporous with cylindrical pores according to IUPAC. The typical regions could be identified in the isotherm of Cys-PMO, which clearly indicates the formation of 2D hexagonal (P6mm) material. The specific area, pore volume, and pore diameter of Cys-PMO hybrid nanoparticles were identified as 691 m2 g−1, 0.59 cm3 g−1, and 3.1 nm, respectively. Cys-PMO hybrid nanoparticles were characterized by low angle XRD in order to analyze the mesostructural order. The XRD pattern of Cys-PMO hybrid nanoparticles (Figure 3a) showed four distinct diffraction patterns corresponding to (100), (110), (200), and (210), which represents the good 2D mesostructure of materials with typical hexagonal P6mm symmetry.23 Further, TGA was performed for Cys-PMO hybrid nanoparticles, as shown in Figure 3b. The weight loss at 100 °C was due to a loss of physisorbed water molecules. The weight loss at 100−450 °C (200−450 °C; 13 wt % for Cys-PMO) is due to the degradation of the Cys-bis-silane precursor organic molecules that are present in the framework structure of Cys-PMO. In addition, thermal degradation between 450 and 800 °C was due to the condensation of silanol functional groups (16 wt %). This result demonstrates that the Cys-bis-silane precursor was successfully incorporated into the framework structure of Cys-PMO. As shown in Figure 4a, SEM images showed clear morphology as a nonaggregated cylindrical shape with a smooth surface. This is mainly due to hydrophilic carboxylic groups and disulfide bonds that could create side-on packing during the formation of Cys-PMO.19 The energy dispersive Xray analyzer (EDX) spectra of Cys-PMO hybrid nanoparticles suggest the existence of S elements in the framework structure of Cys-PMO (Figure 4b,c). Further, the average particle size (nm) was measured as 270 nm through a DLS experiment (Figure S3). The HR-TEM images of Cys-PMO hybrid nanoparticles showed a cylindrical morphology with uniform pores of well-ordered mesoporous hexagonal channels (Figure 5a−c). Further, the elemental distribution as existing in the Cys-PMO hybrid nanoparticles was examined using scanning transmission electron microscopy and an energy dispersive system (STEM/EDS). As shown in Figure 5d, the dark field STEM images also supported HR-TEM results. The selected area of EDS elemental mapping of Cys-PMO hybrid nano-

Figure 1. (a) FTIR, (b) 13C CP MAS, and (c) 29Si MAS NMR spectra of Cys-PMO hybrid nanoparticles.

δ ≈ −89, −100, and −109 ppm were attributed to the Q2 [(SiO) 2 Si-(OH) 2 ], Q 3 [(SiO) 3 Si−OH], and Q 4 [(SiO)4Si−O−Si] species, respectively, which are present in the framework of the Cys-PMO. The presence of a bisilylated organic molecule could be visible due to the presence of two additional peaks at δ ≈ −56 and −65 ppm, which corresponds to the siloxane functional groups in the CysPMO. These peaks were attributed to the existence of isolated terminal (SiO)2(OH)SiC (T2) and cross-linked (SiO)3SiC (T3) functional groups, respectively. Figure 2 represents the N2 adsorption−desorption isotherm and pore size distribution (inset) of Cys-PMO hybrid nanoparticles. The isotherm showed a type IV pattern with steep capillary condensation-evaporation steps in the range P/ D

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 3. (a) XRD pattern and (b) TGA curve of Cys-PMO hybrid nanoparticles.

Figure 4. (a) SEM image and (b,c) corresponding selected area of EDX pattern of Cys-PMO hybrid nanoparticles.

the positive charge of Dox (−NH2 groups in its structure) and the negative charge of MSNs. Thus, a high amount of Dox was loaded into Cys-PMO hybrid nanoparticles due to the greater amount of carboxylic acid functionality, which could complex with the amino groups of Dox.24 As shown in Figure 6a, the Dox was released under the influence of pH and redox potentials. Under tumor conditions (pH 5.5 and redox potentials), the Dox molecules could easily come out due to the pH cleavage of Dox and the degradation of S−S bonds available in the framework structure of Cys-PMO hybrid nanoparticles. Therefore, the ability of a high loading level of Dox and the smart (pH and redox) behavior of Cys-PMO hybrid nanoparticles provide a good opportunity to evaluate their potential for intracellularly degradable targeted drug delivery vehicles in cancer cells. In order to analyze the Dox release in vitro, the release experiment was performed in both an extracellular environment (pH 7.4) and an intracellular cancer cell environment (pH 5.5 with or without DTT (10 mM)) at 37 °C, as shown in Figure 6b. The release profiles showed that the Dox release from Cys-PMO hybrid nanoparticles was dependent on both the extracellular (pH 7.4) and intracellular cancer environment (pH 5.5 and 10 mM DTT). The release of Dox from Cys-PMO hybrid nanoparticles in pH 7.4 PBS was much slower than at pH 5.5 and pH 5.5/10 mM DTT after 48 h of incubation. The % Dox released from CysPMO in PBS, acidic pH 5.5, and pH 5.5/10 mM DTT is about 10, 56, and 89%, respectively, for 48 h of incubation time. The enhanced drug release at pH 5.5 with or without DTT (10 mM) was due to the destabilization of Cys-PMO hybrid nanoparticles under acidic pH and redox conditions.

particles (Figure 5d-1) clearly suggested the uniform distribution of Si, O, C, N, and S elements (Figure 5e−i). Moreover, the existence of the S element further proved the Cys-bis-silane precursor framework in the Cys-PMO material. Dox Loading and in Vitro Release Performance. At pH 7.4, the positively charged Dox (pKa 8.6) can easily be bound to carboxylic functional groups through electrostatic interactions.24 In the present study, the Cys-PMO possesses carboxylic functional groups, which are available in the framework of CysPMO hybrid nanoparticles. The Dox loading efficiency of CysPMO at different concentrations of Dox (2, 5, 10, 15, 20, and 30 mg) in the feed composition was shown in Figure S4. The % DLC was increased with increasing concentration of Dox in the feed and could reach up to 50.6% (15.2 mg of Dox per 30 mg of Cys-PMO) at 20 mg of feed of Dox per 30 mg of Cys-PMO hybrid nanoparticles, and their %EE was measured as 76%. The Dox loading was also supported by the BET nitrogen adsorption/desorption measurement of Dox-loaded Cys-PMO hybrid nanoparticles. As shown in Figure S5, the BET surface area decreased from 691 m2 g−1 to 12.26 m2 g−1 when Dox was introduced to Cys-PMO. The pore volume was calculated according to the BJH model for Dox-loaded Cys-PMO, and the value decreased to 0.024 cm3 g−1. The results demonstrated that the pores of Cys-PMO were blocked with Dox drug. In order to confirm the ability of carboxylic functional groups for the higher amount of Dox into Cys-PMO, further MSNs (without Cys) were also tested for Dox loading capability. The results of Dox loading content and their %EE values are shown in Figure S6. The %DLC of MSNs could reach 32.6% (9.8 mg of Dox per 30 mg of MSNs) due to electrostatic interactions of E

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 5. (a) TEM image, (b,c) magnified TEM images, and (d) STEM image (inset (d-1) selected area of STEM), (e−i) corresponding elemental mapping images (Si, O, C, N, and S) of Cys-PMO hybrid nanoparticles.

In Vitro Cellular Cytotoxicity. The cytotoxicity of pristine Cys-PMO and Dox-loaded Cys-PMO hybrid nanoparticles treated with HeLa cells was evaluated by the Prustoblue assay. The pristine Cys-PMO hybrid nanoparticle treated with HeLa cells showed no significant cytotoxicity, even at a higher amount of Cys-PMO (1000 μg/mL; Figure S7). Dox is commonly used as the anticancer drug in the treatment of a wide range of cancers such as stomach, breast, ovarian, cervical, and lung cancer cells. However, the administration of Dox lacks cancer-targeting ability (due to poor drug distribution) and shows serious side effects to the normal cells. Moreover, the hydrophilic nature of Dox further restricts transport through cellular membranes. Hence, in the present study, pH and redoxresponsive Cys-PMO hybrid nanoparticles were designed for loading and delivery of Dox in order to improve the cell targeting ability without affecting the normal cells. The cytotoxicity is dependent on the concentration of free Dox as well as Dox-loaded Cys-PMO hybrid nanoparticles. The cytotoxicity of Dox and Dox-loaded Cys-PMO hybrid nanoparticles was measured in both extracellular conditions (pH

7.4) and the intracellular cancer cell environment (redox and acidic pH). To study the effect of redox-triggered Dox cytotoxicity, the HeLa cells were pretreated with 10 mM GSH-OEt followed by treatment with Dox-loaded Cys-PMO hybrid nanoparticles. In general, GSH-OEt is not toxic to HeLa cells as previously reported.25,26 As shown in Figure 7, cell viability of 83% was noted for Dox-loaded Cys-PMOs treated with HeLa cells under extracellular conditions (pH 7.4 conditions). It was observed that the IC50 concentration of the free Dox-treated HeLa cells was 0.63 μM. The IC50 of HeLa cells treated with Dox-loaded Cys-PMO hybrid nanoparticles (0.56 μM) at pH 5.5/10 mM GSH-OEt was significantly higher than free Dox (0.63 μM) and Dox-loaded Cys-PMO at pH 5.5 (0.81 μM). An in vitro cytotoxicity analysis showed good agreement with in vitro release data. The results demonstrated excellent anticancer activity of Dox-loaded CysPMO hybrid nanoparticles on HeLa cells under intracellular conditions relative to that of extracellular conditions and could make it a smart drug delivery carrier for pH- and redoxtriggered release of Dox to the HeLa cells. F

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Dox release, the cellular internalization into HeLa cells was evaluated by CLSM images to examine the fluorescence intensity of Dox for a 6-h incubation period. HeLa cell nuclei were counterstained with DAPI. As shown in Figure 8a, the

Figure 8. CLSM images of HeLa cells treated with (a) free Dox and Dox loaded Cys-PMO hybrid nanoparticles at (b) pH 7.4, (c) pH 5.5, and (d) pH 5.5 with 10 mM GSH-OEt for 6 h incubation (nucleus stained with DAPI).

fluorescence intensity of Dox (red) was observed in the nucleus of HeLa cells, which is consistent with earlier reports.27 Further, the fluorescence intensity of Dox-loaded Cys-PMO (red) hybrid nanoparticles observed around the nucleus (Figure 8b) indicates the successful uptake of Cys-PMO hybrid nanoparticles into HeLa cells. More red fluorescence was observed in both the cell nucleus and around the nucleus for Dox-loaded Cys-PMO hybrid nanoparticles under pH 5.5 conditions, which indicates that the Dox molecules had come out from CysPMOs under these conditions. Importantly, the red fluorescence signal was higher for Dox-loaded Cys-PMO hybrid nanoparticles under both pH 5.5 and 10 mM GSH-OEt conditions. This finding shows that the high intensity of Dox fluorescence signals was mainly due to the destabilization of Cys-PMO hybrid nanoparticles. It was demonstrated that Doxloaded Cys-PMO hybrid nanoparticles successfully escaped from the lysosome to the cytosol and were destabilized under these conditions.26 Therefore, the developed Cys-PMO hybrid nanoparticles are promising for loading high Dox levels and suitable for intracellularly triggered delivery to the cancer cells.

Figure 6. (a) Schematic representation of destabilization of Cys-PMO hybrid nanoparticles in response to redox and acidic pH environment and (b) in vitro Dox release profle from Cys-PMO hybrid nanoparticles.

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CONCLUSIONS In summary, we described intracellularly degradable cystamineintegrated periodic mesoporous organo silica nanoparticles for cancer drug delivery. Cystamines containing disulfide bonds were incorporated into pore walls of PMO hybrid nanoparticles to destabilize the intracellular cancer environment. The CysPMO hybrid nanoparticles can load a large amount of Dox (50.6%), avoid the premature drug release at the extracellular level, and improve the cytotoxicity at the intracellular environment for HeLa cells. The results pave the way for

Figure 7. In vitro cytotoxicity of free Dox and Dox loaded Cys-PMO hybrid nanoparticles under various conditions.

In Vitro Cellular Uptake and Dox Distribution. The intracellular distribution of Dox and Dox-loaded Cys-PMO hybrid nanoparticles is strongly dependent on the pH and redox potentials. In order to verify the effective intracellular G

DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering

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cost-effective hybrid PMO nanoparticles possessing the combination of a high drug loading level and improved specific delivery capabilities for intracellular degradable cancer treatment.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00558. Figure S1, 1H NMR spectra of cystamine; Scheme S1, preparation of Cys-Bis silane precursor; Figure S2, 1H NMR spectra of cystamine bis-3-(trimethoxysilyl)propylsuccinic acid (Cys-bis-silane) precursor; Figure S3, DLS average size of Cys-PMO silica nanoparticles; Figure S4, % EE and % DLC for Cys-PMO at various concentrations of Dox (2, 5, 10, 15, 20, and 30 mg); Figure S5, N2 adsorption−desorption isotherm and pore size distribution curve of DOX loaded Cys-PMO hybrid nanoparticles; Figure S6, % EE and % DLC for MSNs at various concentrations of Dox (2, 5, 10, 15, and 20 mg); Figure S7, % cell viability of Cys-PMO silica nanoparticles on HeLa cells with a 48 h incubation period (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +82-53-810-2773. Fax: +82-53-810-4686. *E-mail: [email protected]. Tel.: +82-51-510-2407. Fax: +82-5144331. *E-mail: [email protected]. Tel.: +82-53-810-2773. Fax: +82-53810-4686. ORCID

Kummara Madhusudana Rao: 0000-0002-5350-9981 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.M.R. would like to acknowledge financial support from 2017 Yeungnam University Research Grant. C.-S.H. would like to acknowledge financial support from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, Korea; Acceleration Research Program (NRF2014R1A2A1110054584; NRF-2017R1A2B3012961); Brain Korea 21 Plus Program (21A2013800002). S.S.H. would like to acknowledge Basic Science Research Programme through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2017R1D1A3B03031234).

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REFERENCES

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DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

ACS Biomaterials Science & Engineering (23) Parambadath, S.; Mathew, A.; Barnabas, M. J.; Rao, K. M.; Ha, C. S. Periodic mesoporous organosilica (PMO) containing bridged succinamic acid groups as a nanocarrier for sulfamerazine, sulfadiazine and famotidine: Adsorption and release study. Microporous Mesoporous Mater. 2016, 225, 174−184. (24) Zhu, Q.; Jia, L.; Gao, Z.; Wang, C.; Jiang, H.; Zhang, J.; Dong, L. A Tumor Environment Responsive Doxorubicin-Loaded Nanoparticle for Targeted Cancer Therapy. Mol. Pharmaceutics 2014, 11, 3269− 3278. (25) Khorsand, B.; Lapointe, G.; Brett, C.; Oh, J. K. Intracellular Drug Delivery Nanocarriers of Glutathione-Responsive Degradable Block Copolymers Having Pendant Disulfide Linkages. Biomacromolecules 2013, 14, 2103−2111. (26) Liu, J.; Pang, Y.; Huang, W.; Zhu, Z.; Zhu, X.; Zhou, Y.; Yan, D. Redox-Responsive Polyphosphate Nanosized Assemblies: A Smart Drug Delivery Platform for Cancer Therapy. Biomacromolecules 2011, 12, 2407−2415. (27) Sakai-Kato, K.; Ishikura, K.; Oshima, Y.; Tada, M.; Suzuki, T.; Ishii-Watabe, A.; Yamaguchi, T.; Nishiyama, N.; Kataoka, K.; Kawanishi, T.; Okuda, H. Evaluation of intracellular trafficking and clearance from HeLa cells of doxorubicin-bound block copolymers. Int. J. Pharm. 2012, 423, 401−409.

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DOI: 10.1021/acsbiomaterials.7b00558 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX