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Lethal drug combination: Arsenic loaded multiple drug mesoporous silica for theranostic applications
Colloids and Surfaces B: Biointerfaces 123 (2014) 506–514
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Lethal drug combination: Arsenic loaded multiple drug mesoporous silica for theranostic applications Faheem Muhammad a , Jianyun Zhao b , Nan Wang d , Mingyi Guo c , Aifei Wang a , Liang Chen d , Yingjie Guo b , Qin Li e , Guangshan Zhu a,∗ a State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China b College of Life Science, Jilin University, Changchun 130012, PR China c College of Construction Engineering, Jilin University, Changchun 130026, PR China d Department of Radiology, The First Hospital of Jilin University, Changchun 130012, PR China e Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan, QLD 4111, Australia
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Article history: Received 9 April 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: Arsenic trioxide Controlled release Multiple drugs Mesoporous silica Nanotheranostic
a b s t r a c t Simultaneous delivery of multiple therapeutic agents is of great importance for effective chemotherapy due of its well-known drug synergism and suppression to chemoresistance. We report a new theranostic nanoformulation to shuttle multiple chemotherapeutic agents for successfully exterminating cancer cells. This strategy is based on the fabrication of magnetite doped mesoporous silica nanoparticles (MSNs) in which both internal porous and external surface of MSN are respectively exploited to load two different kinds of cytotoxic cargoes. Notably, an exceptionally high quantity (29%) of poorly hydrophobic drug camptothecin (CPT) is loaded into the nanopores of MSNs; however, in previous reports less than 1% loading efficiency is reported. Following CPT loading in the pores of MSNs, another unconventional but FDA approved arsenic trioxide (ATO) is conjugated onto the surface of nanocomposite via exploiting the thiophilic nature of ATO. Cell inhibition performance of dual drug nanoformulation is significantly higher than single drug formulation, possibly due to additional or synergistic effect, as low as 3 g/ml of double drug nanocarrier were found effective to exterminate cancer cells. Besides drug delivery, the presence of superparamagnetic magnetite nanocrystals additionally empowers this system to be used as a contrast agent in magnetic resonance (MR) imaging for either monitoring diseased tissues or feedback of chemotherapy. We anticipate that the integration of combination therapy with nanotechnology coupled with versatile magnetic manipulation feature may prove a significant step forward toward the development of effective theranostic agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over last two decades, Appreciable advancement in nanomedicine has indeed opened numerous new possibilities to improve diagnosis, therapy and monitoring of various debilitating diseases[1–4]. In relation to cancer, chemotherapeutic drugs ubiquitously pose a serious threat to healthy tissues due to their innate ability to target both tumor and normal cells. In order to overcome these obstacles, Nanoparticle-based carriers can provide us lucrative opportunity via specifically targeting
∗ Corresponding author at: State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China. Tel.: +86 431 85168331. E-mail address: [email protected] (G. Zhu). http://dx.doi.org/10.1016/j.colsurfb.2014.09.046 0927-7765/© 2014 Elsevier B.V. All rights reserved.
cancer cells either through exploiting the leaky vasculature or overexpressed receptors on the surface of cancer cells [5–8]. In addition to drug delivery, nanoparticles also provide an incredible prospect of visualizing and enhancing the images of defected tissues [9]. A great numbers of sophisticated nanobased theranostics platforms, including liposome, polymeric micelle, dendrimers, carbon nanotube, and inorganic nanoparticle have been developed to realize simultaneous diagnosis and therapy [10–16]. Among these nanocarriers, mesoporous silica nanoparticles (MSNs) have been emerged as an effective solid supports in the development of theranostic platform [17–19]. The reason behind its selection lies in its exceptional physicochemical properties, such as enormous surface area, biocompatibility, ready functionalization, controlled and stimuli-responsive drug release. Numerous multifunctional mesoporous silica based systems have, till now, been developed by incorporating diverse kinds of materials in monodispersed silica
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nanoparticles [20]. For instance, Hyeon and coworkers initially reported a facile strategy to obtain multifunctional theranostic nanoparticles by simultaneously incorporating magnetite and quantum dots in the mesoporous shell [21]. The same group later tested the in vivo applicability of improved formulation of MSNs [22]. To achieve targeted specificity, Liong et al. [23] investigated folic acid and dye-doped magnetic MSNs nanocomposite for simultaneous drug delivery, magnetic resonance and fluorescence imaging of cancer cells. Lin Jun group similarly reported a series of mesoporous silica based multifunctional nanoparticles [24]. Despite these noticeable developments in MSNs theranostics, it has rarely been explored to concurrently deliver multiple drugs for combination chemotherapy. Combination chemotherapy has long been an effective regimen in clinics, the biomedical experts nowadays are earnestly contemplating on the option to transport multiple therapeutic agents through a single nanovehicle for potentially enhancing drug efficacy and overpowering the cancer chemoresistance [25]. Pioneering work in this regard was carried out by Agrawal et al. [26] who employed liposomal single nanoparticle to deliver 6-mercaptopurine and daunorubicin against Jurkat and Hut 76 T-cell. In another effort, four times higher therapeutic efficacy was reported, using doxorubicin and verapamil co-encapsulating liposomal nanoparticles [27]. Polymer nanoparticles were also explored for combinatorial chemotherapy to transport verapamil and vincristine [28]. But, mesoporous silica has rarely been used to deliver multiple drugs at the same time. On another front, apart from typical anticancer drugs, arsenicbased compounds have been garnering a lot of attention due to their well-proven antitumor potential [29]. Arsenic and its methylated species are generally recognized as carcinogen, but on the other hand, they have also been used as therapeutic agents “Delicious Poison” for thousands of years. Specifically, it has been extensively used in Chinese medicine to treat ulcers plague, malaria, cancer, syphilis, and rheumatisms. In recent times, Dr. Thomas Fowler introduced Fowlers solution (As2 O3 in potassium bicarbonate solution) to treat various malignant diseases such as leukemia, Hodgkin’s disease, as well as non-malignant diseases including eczema, asthma, pemphigus, and psoriasis. Recently, A clinical trial was carried based on the principal of traditional Chinese medicine “using a toxic agent against a toxic agent” to treat acute promyelocytic leukemia (APL). Extremely encouraging results (85% remission rate) rekindled the interest in arsenicals and now it is being used as a frontline agent to tackle hematologic malignancies. The therapeutic efficiency of ATO is ascribed to the degradation of an oncogenic protein that drives the growth of APL cells, PML–RAR ˛ [30]. Besides fluid cancers, ATO has also been investigated against several solid tumors, including gastric cancer, esophageal carcinoma, neuroblastoma liver, ovarian, cervical, breast, prostate, and head and neck cancers [31,32]. Due to poor pharmacokinetics and dose-limiting toxicity, much higher As2 O3 dosages are required for solid tumors which restrict its utility in solid tumors. Various nanoparticulate formulations of ATO have been fabricated to enhance the efficacy and mitigate the toxicity of ATO. For example, ATO has been encapsulated in liposomal or polymeric vesicles but unfortunately large amount of drug is lost within hours at physiological condition [33]. These challenges intensify the need to develop more robust drug nanocarriers for delivering multiple toxic drugs in targeted fashion. In order to meet these challenges, herein, we report a straightforward strategy to fabricate magnetic and dual drug-containing MSNs for simultaneously shuttling drugs cocktail to the site of action. Firstly magnetite nanoparticles were tethered onto the MSNs surface via EDC chemistry. Porous structure of MSNs was then exploited to load significantly lethal dose of campothetacin (CPT) into the nanochannels, afterwards, ATO was tethered onto thiol functionalized exterior surface of MSNs. Prominently,
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the presence of magnetic particles can enable to achieve not only magnetic targeted delivery of cytotoxic compounds to intended site in the body but also offers a simultaneous opportunity to monitor the therapeutic response of therapy via magnetic resonance imaging (MRI). 2. Experimental 2.1. Materials Chemical reagents used in this study are of analytical grade and used as received. Cetyltrimethylammonium bromide (CTAB), 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich. 3-Mercaptopropyltriethoxysilane (MPTES), 3-aminopropyltriethoxysilane (APTES), mercaptopropionic acid (MPA), camptothecin (CPT), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), tetraethyl orthosilicate (TEOS, 99.98%), absolute ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from Aladdin reagent company. 2.2. Synthesis of citrate capped magnetite Following a literature procedure, citric acid modified magnetite nanocrystals were prepared via co-precipitation method. Typically, 0.86 g FeCl2 and 2.35 g FeCl3 were dissolved in 40 mL distilled water and heated to 80 ◦ C under argon in a three-necked flask. Under vigorous stirring the, 5 mL of NH4 OH was added into reaction mixture by syringe and the heating continued for 30 min. Next, 1 g of citric acid solution (2 mL solution) was introduced and temperature was raised to 95 ◦ C. Stirring was continued for an extra 90 min to complete the coating on the surface of magnetite. The resulting black precipitated magnetite nanoparticles were later washed with excess of ethanol thrice by magnetic separation. Finally, precipitate was re-dispersed in water to obtain transparent solution of well dispersed nanoparticles. 2.3. Synthesis of amine functionalized mesoporous silica Mesoporous material was prepared by following a previously described method. Typically; 0.5 g of CTAB was first dissolved in 240 mL deionized water. Sodium hydroxide aqueous solution (2 M, 1.8 mL) was added into CTAB solution and then the reaction temperature was raised to 80 ◦ C. After stabilizing the temperature, TEOS (2.5 mL, 11.2 mmol) and 250 L APTES were consecutively added dropwise to the surfactant solution and held the reaction mixture for 2 h to give a white precipitate. The solid product was filtered, washed with deionized water and ethanol, dried at 60 ◦ C to yield the as-synthesized amine functionalized MSNPs. To maximize the anchoring of amine group, as-synthesized MSNs were refluxed for 12 h in 20 mL anhydrous toluene (300 L APTES). The product was recovered by centrifugation and washed with ethanol trice. Finally, surfactant (CTAB) was removed by refluxing the nanoparticle in acidic methanolic solution for 6 h. 2.4. Magnetite conjugation and mercapto functionalization of mesoporous silica nanoparticles Magnetite nanoparticles were anchored onto the surface of MSNs via EDC chemistry. Amine functionalized MSNs (200 mg) were dispersed in 20 mL of water. On the other hand, magnetite NPs (20 mg) were dispersed in 10 mL of water in another beaker and activated by EDC (5 mg). Both solutions were mixed to obtain magnetite conjugated MSNs (Fe3 O4 @MSNs). The solid product was then filtered, washed with deionized water and ethanol. In order to modify the remaining amine group into thiol moiety for ATO
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capturing, Fe3 O4 @MSNs nanocomposite (100 mg) was dispersed into water, and 5 mL of EDC activated MPA solution (20 L) was added into above solution. After stirring for 10 min, the product was recovered by centrifugation and washed with ethanol. 2.5. Loading of both drugs and release experiments First, ATO was immobilized onto the surface of thiol functionalized Fe3 O4 @MSNs. Thiol functionalized sample (60 mg) was dispersed into 20 mL water. Meanwhile, 50 mg of arsenic trioxide (As(OH)3 ) was dissolved in small quantity of 2 M NaOH solution and pH of the solution was adjusted to 8.0 in order to avoid silica dissolution. Equivalent amount of ATO solution was added into above Fe3 O4 @MSNs solution at room temperature. After 30 min stirring both solutions were centrifuged and washed with water. For loading hydrophobic camptothecin (CPT) in the nanochannels of mesoporous channels, ATO–Fe3 O4 @MSNs (50 mg) was introduced to DMSO solution of camptothecin (5 mL, 3 mg/mL) and stirred for 12 h at 25 ◦ C. After stirring in DMSO, the solution was transferred into 30 mL water and stirred for further 6 h. CPT was also similarly loaded in MSN nanoparticles devoid of ATO. The solution was then centrifuged and determined the amount of CPT loading by using UV/visible spectroscopy. Camptothecin and ATO release were carried out by soaking the CPT–ATO–Fe3 O4 @MSNs formulation in phosphate buffer (pH 7.4) and a dialysis bag diffusion technique was used. Briefly, 3.5 mg of samples was first dispersed in 5 mL of buffer and sealed in a dialysis bag (molecular weight cutoff = 8000). The dialysis bag was then submerged in 25 mL of the same buffer solution and stirred for five days. The released CPT in the buffer was collected at predetermined time intervals and analyzed by UV/vis spectroscopy using 365 nm wavelengths. On the other hand, ICP-OES was used to evaluate the release of arsenic. 2.6. Cell culture Pancreatic cancer cells (BxPC-3) were grown in monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Tianhang bioreagent Co., Zhejiang) and penicillin/streptomycin (100 U mL−1 and 100 g/ml, respectively) in a humidified 5% CO2 atmosphere at 37 ◦ C. 2.7. Cell viability (MTT assay) The viability of cells in the presence of nanoparticles was investigated using 3-[4,5-dimethylthialzol-2-yl]-2,5diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was carried out in triplicate in the following manner. For MTT assay, BxPC-3 were seeded into 96-well plates at a density of 8 × 103 per well in 100 L of media and grown overnight. The cells were then incubated with various Fe3 O4 @MSNs, ATOFe3 O4 @MSNs, CPT–ATO–Fe3 O4 @MSNs, and free CPT for 72 h. Afterwards, cells were incubated in media containing 0.5 mg/mL of MTT for 4 h. The precipitated formazan violet crystals were dissolved in 100 L of 10% SDS in 10 mmol HCl solution at 37 ◦ C overnight. The absorbance was measured at 570 nm by multidetection microplate reader (SynergyTM HT, BioTek Instruments Inc., USA). 2.8. Confocal laser scanning microscopy (CLSM) To check cellular uptake and CPT release, BxPC-3 were cultured in an 12-well chamber slide with one piece of cover glass at the bottom of each chamber in incubation medium (DMEM) for 4 h. CPT–ATO–Fe3 O4 @MSNs was added into the incubation medium at the concentration of 50 and 100 g/ml for 3 h incubation in 5% CO2 at 37 ◦ C. After the medium was removed, the cells were
washed twice with PBS (pH = 7.4) and the cover glass was visualized under a laser scanning confocal microscope (FluoView FV1000, Olympus). 2.9. Characterization The powder XRD patterns were recorded on a Rigaku D/Max ˚ The 2550 X-ray diffractometer with Cu-K␣ radiation ( = 1.5418 A). morphologies and detailed structure of the samples were recorded using JEOL JSM-6700F field-emission scanning electron microscope (SEM) and FEI Tecnai G2 F20 S-TWIN transmission electron micro˚ Elemental analysis was carried out on scope (TEM) ( = 1.5418 A). Perkin–Elmer ICP-OES Optima 3300DV. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer. The nitrogen adsorption and desorption isotherms were measured at liquid N2 temperature by using a Quantachrom Autosorb-iQ after the sample was degassed for 12 h at 150 ◦ C. Surface area was calculated according to the conventional BET method and the adsorption branches of the isotherms were used for the calculation of the pore parameters using the BJH method 3. Results and discussions 3.1. Characterization of multiple drug carrying theranostic nanoconstructs (ATO–CPT–Fe3 O4 @MSNs) Citric acid stabilized magnetite and MCM-41 type mesoporous silica nanoparticles were firstly prepared according to previously reported methods. The surface of mesoporous silica was functionalized with amine group via both co-condensation and post-grafting procedures to maximize the amount of amine moiety. After surfactant removal, the resulting citric acid capped magnetite nanoparticles were conjugated onto the surface of mesoporous silica particles (Fe3 O4 @MSNs) using EDC activation chemistry. In order to efficiently load ATO, the surfaces of Fe3 O4 @MSNs was functionalized with mercapto groups. Use of mercaptopropyltriethoxysilane (MPTS), as a thiol introducing agent, invariably resulted in an enhanced hydrophobicity of MSN nanoparticles, thus reducing the water dispersibility of nanocarriers. In order to avoid this restraint, mercaptopropionic acid (MPA) was used to introduce thiol functionality using EDC chemistry by reacting the amine functionalized Fe3 O4 @MSNs with carboxyl group of MPA. Finally, ATO was tethered onto the surface of Fe3 O4 @MSNs [34,35], and camptothecin (a hydrophobic topoisomerase I inhibitor) was loaded into the empty nanochannels of composite. Using UV/vis absorption measurements, loaded amount of drug was calculated to be 290 mg of CPT per gram of Fe3O4@MSNs. This exceptionally high loading of poorly water soluble CPT can be highly advantageous to improve the in vivo efficacy of this promising drug. SEM (Fig. 1a) and TEM images (Fig. 1b) of Fe3 O4 @MSNs indicates monodispersed and sphere-shaped mesoporous silica nanoparticles with a diameter of ∼100 nm. The obtained size is considered really suitable for intracellular uptake and subsequent drug release applications. The existence of 2D hexagonal porous structure with empty channels (2–3 nm) provides enough space to encapsulate relatively large amounts of therapeutic molecules. Moreover, both SEM and TEM images also verify the presence of magnetite nanocrystals, as vivid black spots onto the surface of MSNs can be clearly seen. In this study, heavy metals affinity toward thiol group was exploited for immobilizing antitumor drug ATO. It is worth mentioning that even magnetite (Fe3 O4 ) nanoparticles have previously been demonstrated as potential sorbents for arsenic removal in drinking water [36,37]. Hence, the presence of magnetite nanoparticles onto the surface of MSNs also provided an added opportunity to enhance the ATO drug loading by capitalizing
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Fig. 1. (a) SEM and (b) TEM micrographs of As–Fe3 O4 @MSNs. (c, d) High resolution TEM and HAADF-STEM survey images which indicate the existence of As, Fe, Si, and O in As–Fe3 O4 @MSNs nanoformulation.
the affinity of magnetite for heavy metals. To confirm the immobilization of ATO onto Fe3 O4 @MSNs surface, elemental analysis was carried by using energy dispersive X-ray spectroscopy (EDX) and mapping. Compositional analysis of nanocomposite was carried out
using EDX mapping (Fig. 1c and d) which confirms the presence of elemental signal of iron, arsenic and sulfur. In addition to these signals, silicon and oxygen signals (SiO2 ) are also observed in mapping. In addition to mapping, arsenic and iron signals can also be
Fig. 2. (a) Nitrogen adsorption–desorption isotherms of Fe3 O4 @MSNs. (b) EDX spectrum of As–Fe3 O4 @MSNs. (c) Wide-angle X-ray diffraction (XRD) patterns of Fe3 O4 (black) and Fe3 O4 @MSNs (red). (d) FTIR spectra of MSNs-NH2 , As–Fe3 O4 @MSNs, CPT–Fe3 O4 @MSNs and Fe3 O4 –CA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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thiol moiety is also detected in the form of sulfur signal in the XPS analysis. The existence of SH groups in Fe3 O4 @MSNs nanocomposite is obvious from photoemission peaks of the 2p orbitals of sulfur in the SH-Fe3 O4 @MSNs spectrum at 164.0 eV (Fig. 3d), Arsenic displays an intense XPS line, As 3d, at 44.4 eV which confirms the immobilization of ATO onto Fe3 O4 @MSNs for therapeutic purposes (Fig. 3c). XPS analysis similarly provided a proof for the existence of iron with a peak at electron binding energy of 711.1 eV. 3.2. Drug release profiles of multiple drugs
Scheme 1. Schematic presentation of synthetic and loading strategies to encapsulate and tether drug cocktail molecules in magnetic porous architecture.
seen in TEM-associated EDX spectrum, suggesting the magnetite presence and conjugation of ATO in the resulting nanocomposites (Fig. 2b). Nitrogen sorption–desorption results of MSNs displayed a type IV isotherm with a hysteresis loop, indicating the existence of mesoporous structure. The mesopores size distribution indicates an intense peak placed at the mean value of 2.4 nm, implying a uniform mesoporous structure. But after magnetite conjugation, the size of hysteresis loop is decreased, as shown in Fig. 2a. Surface area (BET) of nanocomposite was also reduced to 728 from 884 m2 g−1 . Such porous architecture renders mesoporous silica as an ideal candidate for loading large quantities cytotoxicdrugs (Scheme 1). Powder X-ray diffraction was carried out to determine the purity of magnetic particles, the obtained XRD patterns (Fig. 2c) can easily be indexed to magnetite phase, whereas the magnetite conjugated MSNs nanoformulation shows similar diffraction peaks but of relatively low intensity due to the existence of silica matrix. Fourier transform infrared (FTIR) spectroscopy verified the nanoparticles conjugation and drug loading processes (Fig. 2d). Chemisorption of citric acid onto magnetite occurs through the COOH interaction. As an indication, the characteristic 1715 cm−1 peak of citric acid (C O vibration) shifts to 1618 cm−1 in citric acid passivated magnetite, suggesting the formation of complex between Fe of the magnetite surface and citric acid. This shifts or chemisorption imparts C O a partial single bond character. Moreover, a band at ∼1400 cm−1 can be assigned to the asymmetric stretching of CO from COOH moiety. Low-intensity bands between 400 and 600 cm−1 are ascribed to stretching and torsional vibration modes of magnetite. Amine functionalization of MSNs is established by the presence of bands at 3446 and 1640 cm−1 which indicates the N H stretching vibration and NH2 bending mode of free NH2 groups respectively. The appearance of bands between 2800 and 3000 cm−1 is ascribed to C H stretching vibrations of propyl group of APTES, as shown in Fig. 2d. Appearance of additional peaks in CPT loaded nanoformulation validates the successful loading of CPT. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) evaluated the quantity of arsenic in double drug nanoformulation. The concentration of arsenic was found to be 35 g per 1 mg of Fe3 O4 @MSNs. X-ray photoelectron spectroscopy (XPS) is a powerful tool to characterize the surface of a materials with a depth detection limit of about 5–10 nm, therefore, we employed this analytical technique to analyze the outermost layers of the MSNs composite to determine the elemental composition of drug loaded samples. In the survey spectrum of the ATO loaded Fe3 O4 @MSNs (Fig. 3), O, Si, Fe and arsenic signals are expectedly detected. Additionally, a small amount of carbon, nitrogen and sulfur are also detected. The C signal is basically attributed to the propyl moieties of triethoxysilane and mercaptopropionic acid. ATO capturing
In vitro release experiments were carried out to assess the release of loaded CPT from CPT–Fe3 O4 @MSN nanoformulation, under somewhat physiological conditions in PBS at pH 7.4 and at 25 ◦ C. Under these conditions, hydrophobic CPT release kinetics can be assumed to be controlled by diffusion since no appreciable degradation of mesoporous silica occurs at pH 7.4. Expectedly, the release profile (Fig. 4a) displays a controlled release pattern of anticancer drug CPT from mesoporous nanochannels which is consistent with previous reports, drug release was monitored for 5 days in this experiment [38]. In contrast to previous studies, this report achieved an exceptionally high loading of CPT (29%) in the nanochannels of porous silica. Such a high loading of hydrophobic drug in inorganic nanocarrier almost competes with various prodrug nanoformulations. Metal ions are soft acids and have strong affinity toward soft bases such as thiol groups. According to Hard/Soft Acid/Base (HSAB) theory, thiol functionalized outer surface of mesoporous silica binds with arsenic (ATO) to make a highly stable metal thiolates like sulfide minerals. Thiol arsenic bond is so stable; but a slight ATO release (10%) over the period of three days from ATO-Fe3 O4 @MSNs (Fig. 4b) points out to another important aspect which is the stability of silica nanocarrier. It is well-known that silica is slowly etched in PBS buffer (pH = 7.4), as ATO is attached onto the surface of MSNs, that is why, silica etching resulted in a slow release of chemotherapeutic agent (ATO) over a specified period of time. ICPOES results indicate that approximately 10% arsenic is untethered from the surface of MSNs. 3.3. Magnetic attributes and MRI study Besides just pinpointed delivery of drugs to diseased sites, the focus of biomedical researchers is nowadays to develop such nanoplatforms which not only ferry drugs to targeted site but also perform imaging to realize diagnosis and subsequent monitoring of therapeutic feedback. As a result, a new biomedical sub-discipline, known as theranostics, has been emerged to integrate both therapeutic and diagnostic functionalities into one nanoparticle. Magnetite nanoparticles were thus introduced to this dual drug carrying nanoparticle to serve as a theranostic agent. Magnetic hysteresis curves of both Fe3 O4 and Fe3 O4 @MSNs were acquired at room temperature using vibrating sample magnetometer, as demonstrated in Fig. 5. The magnetization curve of citric acid capped magnetite and Fe3 O4 @MSNs nanocomposite showed no hysteresis which indicates the superparamagnetic character of both nanoparticles. Saturation magnetization (Fig. 5a) of citric acid coated material was calculated to be around 80 emu g−1 . Reduction in the magnetization from 80 emu g−1 to 7 emu g−1 was observed in case of mesoporous silica-magnetite nanocomposite due to addition of higher mass fraction of silica content. Iron oxide nanocrystals are being widely utilized as contrast agents in MRI due to their negative enhancement effect on T2 -weighted sequences [39,40]. To assess the applicability of magnetite containing MSNs as MRI contrast agent, different concentrations of Fe3 O4 @MSNs were suspended in 1% agarose gel (4 mg/mL) and
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Fig. 3. (a) XPS survey spectrum of As-Fe3 O4 @MSNs, (b) Fe 2p3/2 , 2p1/2 peak, (c) As 3d and (d) S 2p.
placed in the centrifuge tubes. The MRI images of mere water and Fe3 O4 @MSNs containing tubes can be seen in Fig. 5c. T2 -weighted MR images of Fe3 O4 @MSNs samples appeared dark with increasing iron concentration due to the decrease in T2 relaxation. In contrast, tube having water remained bright and indistinguishable. MRI contrast agent performance is assessed on the basis of their
relaxivity or the alteration in the relaxation time of water protons in the presence of the agent per unit concentration. The relaxivity (r2 ) of Fe3 O4 @MSNs was found to be 161 mM−1 s−1 and can be seen in Fig. 5b. Mathematically, the r2 value of Fe3 O4 @MSNs was determined by calculating the slope of a plot of 1/T2 versus Fe concentration.
Fig. 4. (a) Release profile of camptothecin from CPT–As–Fe3 O4 @MSNs nanoformulation at pH 7.4. (b) Release profile of ATO from CPT–As–Fe3 O4 @MSNs at pH 7.4 and 25 ◦ C. The release of drug molecules was monitored by UV–vis spectrophotometer (365 nm) and inductively coupled plasma-atomic emission spectrometry (ICP-AES).
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Fig. 5. (a) M–H curves of Fe3 O4 (black line) and Fe3 O4 @MSNs (red line). Inset: photographs display water dispersibility and quick magnetophoretic separation of Fe3 O4 @MSNs nanocomposite. (b) T2 relaxation rate plots (1/T2 ) versus Fe concentration. (c) T2 -weighted MR images of dispersed Fe3 O4 @MSNs at different Fe concentrations in 1.0% agarose. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Cellular uptake, intracellular drug release and cell viability assay Confocal laser scanning microscopy (CLSM) was employed to investigate the cellular uptake and subsequent intracellular drug release from Fe3 O4 @MSN formulations (Fig. 6). Predetermined concentrations of CPT loaded Fe3 O4 @MSNs formulation was incubated with pancreatic cancer cells. Following 4 h incubation, the fluorescence micrographs clearly indicate that Fe3 O4 @MSNs nanoparticles are spontaneously internalized into BxPC-3cells. Manifestation of blue fluorescence in cytoplasmic region verified the intracellular release of CPT from ATO–CPT–Fe3 O4 @MSNs formulation as depicted in in vitro release data. Anticancer efficacy of different Fe3 O4 @MSNs formulations was tested using same pancreatic
cancer cell line (BxPC-3). Cells were treated with different concentrations of nanoformulations for 72 h, and cytotoxicity was assessed by MTT analysis. Although, the biocompatibility of magnetite and mesoporous silica is extensively reported, but we still quantified the cytotoxic potency of this composite and drugs containing Fe3 O4 @MSNs formulations. As mentioned above, arsenic and its methylated derivative are ordinarily recognized as carcinogens but now it has also been approved by FDA as an effective antileukaemic agent [41]. Extensive research has been carried out to extend the antineoplastic property of arsenic-based drugs to solid tumors. To date encouraging results about inhibiting growth and inducing apoptosis in human solid cancer cells, at clinically achievable concentrations, have been reported. Notwithstanding, its indiscriminate action and dose-dependent toxicity constrain utility of
Fig. 6. Confocal laser scanning microscopy images of BxPC-3 cells after 4 h incubation with CPT–As–Fe3 O4 @MSNs nanoformulation (50 g/ml). (a) Bright field image, (b) fluorescent image and (c) merged image. Blue fluorescence indicates the cellular uptake of and subsequent release of camptothecin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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intracellular uptake and excellent therapeutic response in cancer cell. Exposure of ATO carrying nanoformulation effectively inhibited the growth of solid tumor cells; however, simultaneous delivery of two drugs via single nanoparticle exhibited a more pronounced cytotoxic effect probably due to drugs synergism. Merely 3 g/ml of double drug nanocarrier was found effective to exterminate tumor cells. These results advocate that the resulting magnetic nanococktail could be a promising addition in the inventory of theranostic to mitigate the severe side effects of toxic drugs on normal cells and simultaneously monitor the efficacy of chemotherapy.
Acknowledgements
Fig. 7. In vitro viability of BxPC-3 cells in the presence of different concentrations of Fe3 O4 @MSNs, As–Fe3 O4 @MSNs, CPT–Fe3 O4 @MSNs and CPT–As–Fe3 O4 @MSNs, the incubation time was 72 h.
We are grateful to the financial support from National Basic Research Program of China (973 Program, grant no. 2012CB821700), Major International (Regional) Joint Research Project of NSFC (grant no. 21120102034) and NSFC (grant no. 20831002).
References such a promising anticancer agent in solid tumors. To tackle these problems, we loaded ATO onto porous magnetic nanoparticles to specifically target cancer cells via exploiting passive and magnetic targeting approach. In vitro efficacy of ATO formulation at different concentrations was tested against BxPC-3 cells. ATO containing nanocarrier exhibited a notable killing efficiency. These results indicate that as little as 6 g/ml amount of ATO–Fe3 O4 @MSNs is enough to kill almost 50% of the cancer cells as shown in Fig. 7. As far as two drug formulations are concerned, significantly high amount of hydrophobic drug (CPT) was loaded into the channels of ATO–Fe3 O4 @MSNs, as a model drug, for ensuring efficient and controlled release of this highly promising anticancer drug, otherwise poor water solubility mars its excellent potential. This study reports a substantial amount of CPT loading (290 mg/g) compared to previously published studies, therefore, higher cell inhibition efficiency is expected in case of two drugs formulation. Anticipatedly, two drugs formulation (ATO–CPT–Fe3 O4 @MSNs) exhibits the greatest potency to inhibit the growth of BxPC-3 cell lines compared to other formulation at the same dose. Prominently, as low as 3 g/ml MSN double drug nanoformulation enabled the inhibition of almost 75% of the cancer cells. This enhanced potency can be attributed to different intracellular route for CPT; otherwise hydrophobic drug (CPT) is administered in DMSO and got precipitated in case of aqueous condition. In addition, drug synergism can also be considered a major factor for this enhanced cytotoxicity. It should also be kept in mind that two drugs cocktail not only target two different mechanisms to fight cancer but can also avoid multi drug resistance which is a major cause of chemotherapy failure due to well established mechanism to bypass resistance through endocytosis route. Based on these findings, the double drug magnetic nanoformulation can potentially be used as a platform to deliver multiple highly cytotoxic drugs specifically to cancer cells.
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In conclusion, we have engineered a multifunctional mesoporous silica based nanocarrier to unify clinically often-prescribed combinational chemotherapy with nanotechnology. Novelty of this system lies in the exploitation of both external and internal regions of mesoporous silica to load two dissimilar cytotoxic drugs by following different approaches. In addition to drug ferrying capabilities, the resulting nanosystem also carries magnetic nanoparticles which impart magnetic manipulation and MRI diagnosis functionalities. MTT and confocal microscopy studies confirmed the
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Lethal drug combination: Arsenic loaded multiple drug mesoporous silica for theranostic applications Faheem Muhammad a , Jianyun Zhao b , Nan Wang d , Mingyi Guo c , Aifei Wang a , Liang Chen d , Yingjie Guo b , Qin Li e , Guangshan Zhu a,∗ a State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China b College of Life Science, Jilin University, Changchun 130012, PR China c College of Construction Engineering, Jilin University, Changchun 130026, PR China d Department of Radiology, The First Hospital of Jilin University, Changchun 130012, PR China e Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan, QLD 4111, Australia
a r t i c l e
i n f o
Article history: Received 9 April 2014 Received in revised form 22 September 2014 Accepted 23 September 2014 Available online 2 October 2014 Keywords: Arsenic trioxide Controlled release Multiple drugs Mesoporous silica Nanotheranostic
a b s t r a c t Simultaneous delivery of multiple therapeutic agents is of great importance for effective chemotherapy due of its well-known drug synergism and suppression to chemoresistance. We report a new theranostic nanoformulation to shuttle multiple chemotherapeutic agents for successfully exterminating cancer cells. This strategy is based on the fabrication of magnetite doped mesoporous silica nanoparticles (MSNs) in which both internal porous and external surface of MSN are respectively exploited to load two different kinds of cytotoxic cargoes. Notably, an exceptionally high quantity (29%) of poorly hydrophobic drug camptothecin (CPT) is loaded into the nanopores of MSNs; however, in previous reports less than 1% loading efficiency is reported. Following CPT loading in the pores of MSNs, another unconventional but FDA approved arsenic trioxide (ATO) is conjugated onto the surface of nanocomposite via exploiting the thiophilic nature of ATO. Cell inhibition performance of dual drug nanoformulation is significantly higher than single drug formulation, possibly due to additional or synergistic effect, as low as 3 g/ml of double drug nanocarrier were found effective to exterminate cancer cells. Besides drug delivery, the presence of superparamagnetic magnetite nanocrystals additionally empowers this system to be used as a contrast agent in magnetic resonance (MR) imaging for either monitoring diseased tissues or feedback of chemotherapy. We anticipate that the integration of combination therapy with nanotechnology coupled with versatile magnetic manipulation feature may prove a significant step forward toward the development of effective theranostic agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Over last two decades, Appreciable advancement in nanomedicine has indeed opened numerous new possibilities to improve diagnosis, therapy and monitoring of various debilitating diseases[1–4]. In relation to cancer, chemotherapeutic drugs ubiquitously pose a serious threat to healthy tissues due to their innate ability to target both tumor and normal cells. In order to overcome these obstacles, Nanoparticle-based carriers can provide us lucrative opportunity via specifically targeting
∗ Corresponding author at: State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China. Tel.: +86 431 85168331. E-mail address: [email protected] (G. Zhu). http://dx.doi.org/10.1016/j.colsurfb.2014.09.046 0927-7765/© 2014 Elsevier B.V. All rights reserved.
cancer cells either through exploiting the leaky vasculature or overexpressed receptors on the surface of cancer cells [5–8]. In addition to drug delivery, nanoparticles also provide an incredible prospect of visualizing and enhancing the images of defected tissues [9]. A great numbers of sophisticated nanobased theranostics platforms, including liposome, polymeric micelle, dendrimers, carbon nanotube, and inorganic nanoparticle have been developed to realize simultaneous diagnosis and therapy [10–16]. Among these nanocarriers, mesoporous silica nanoparticles (MSNs) have been emerged as an effective solid supports in the development of theranostic platform [17–19]. The reason behind its selection lies in its exceptional physicochemical properties, such as enormous surface area, biocompatibility, ready functionalization, controlled and stimuli-responsive drug release. Numerous multifunctional mesoporous silica based systems have, till now, been developed by incorporating diverse kinds of materials in monodispersed silica
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nanoparticles [20]. For instance, Hyeon and coworkers initially reported a facile strategy to obtain multifunctional theranostic nanoparticles by simultaneously incorporating magnetite and quantum dots in the mesoporous shell [21]. The same group later tested the in vivo applicability of improved formulation of MSNs [22]. To achieve targeted specificity, Liong et al. [23] investigated folic acid and dye-doped magnetic MSNs nanocomposite for simultaneous drug delivery, magnetic resonance and fluorescence imaging of cancer cells. Lin Jun group similarly reported a series of mesoporous silica based multifunctional nanoparticles [24]. Despite these noticeable developments in MSNs theranostics, it has rarely been explored to concurrently deliver multiple drugs for combination chemotherapy. Combination chemotherapy has long been an effective regimen in clinics, the biomedical experts nowadays are earnestly contemplating on the option to transport multiple therapeutic agents through a single nanovehicle for potentially enhancing drug efficacy and overpowering the cancer chemoresistance [25]. Pioneering work in this regard was carried out by Agrawal et al. [26] who employed liposomal single nanoparticle to deliver 6-mercaptopurine and daunorubicin against Jurkat and Hut 76 T-cell. In another effort, four times higher therapeutic efficacy was reported, using doxorubicin and verapamil co-encapsulating liposomal nanoparticles [27]. Polymer nanoparticles were also explored for combinatorial chemotherapy to transport verapamil and vincristine [28]. But, mesoporous silica has rarely been used to deliver multiple drugs at the same time. On another front, apart from typical anticancer drugs, arsenicbased compounds have been garnering a lot of attention due to their well-proven antitumor potential [29]. Arsenic and its methylated species are generally recognized as carcinogen, but on the other hand, they have also been used as therapeutic agents “Delicious Poison” for thousands of years. Specifically, it has been extensively used in Chinese medicine to treat ulcers plague, malaria, cancer, syphilis, and rheumatisms. In recent times, Dr. Thomas Fowler introduced Fowlers solution (As2 O3 in potassium bicarbonate solution) to treat various malignant diseases such as leukemia, Hodgkin’s disease, as well as non-malignant diseases including eczema, asthma, pemphigus, and psoriasis. Recently, A clinical trial was carried based on the principal of traditional Chinese medicine “using a toxic agent against a toxic agent” to treat acute promyelocytic leukemia (APL). Extremely encouraging results (85% remission rate) rekindled the interest in arsenicals and now it is being used as a frontline agent to tackle hematologic malignancies. The therapeutic efficiency of ATO is ascribed to the degradation of an oncogenic protein that drives the growth of APL cells, PML–RAR ˛ [30]. Besides fluid cancers, ATO has also been investigated against several solid tumors, including gastric cancer, esophageal carcinoma, neuroblastoma liver, ovarian, cervical, breast, prostate, and head and neck cancers [31,32]. Due to poor pharmacokinetics and dose-limiting toxicity, much higher As2 O3 dosages are required for solid tumors which restrict its utility in solid tumors. Various nanoparticulate formulations of ATO have been fabricated to enhance the efficacy and mitigate the toxicity of ATO. For example, ATO has been encapsulated in liposomal or polymeric vesicles but unfortunately large amount of drug is lost within hours at physiological condition [33]. These challenges intensify the need to develop more robust drug nanocarriers for delivering multiple toxic drugs in targeted fashion. In order to meet these challenges, herein, we report a straightforward strategy to fabricate magnetic and dual drug-containing MSNs for simultaneously shuttling drugs cocktail to the site of action. Firstly magnetite nanoparticles were tethered onto the MSNs surface via EDC chemistry. Porous structure of MSNs was then exploited to load significantly lethal dose of campothetacin (CPT) into the nanochannels, afterwards, ATO was tethered onto thiol functionalized exterior surface of MSNs. Prominently,
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the presence of magnetic particles can enable to achieve not only magnetic targeted delivery of cytotoxic compounds to intended site in the body but also offers a simultaneous opportunity to monitor the therapeutic response of therapy via magnetic resonance imaging (MRI). 2. Experimental 2.1. Materials Chemical reagents used in this study are of analytical grade and used as received. Cetyltrimethylammonium bromide (CTAB), 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich. 3-Mercaptopropyltriethoxysilane (MPTES), 3-aminopropyltriethoxysilane (APTES), mercaptopropionic acid (MPA), camptothecin (CPT), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), tetraethyl orthosilicate (TEOS, 99.98%), absolute ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from Aladdin reagent company. 2.2. Synthesis of citrate capped magnetite Following a literature procedure, citric acid modified magnetite nanocrystals were prepared via co-precipitation method. Typically, 0.86 g FeCl2 and 2.35 g FeCl3 were dissolved in 40 mL distilled water and heated to 80 ◦ C under argon in a three-necked flask. Under vigorous stirring the, 5 mL of NH4 OH was added into reaction mixture by syringe and the heating continued for 30 min. Next, 1 g of citric acid solution (2 mL solution) was introduced and temperature was raised to 95 ◦ C. Stirring was continued for an extra 90 min to complete the coating on the surface of magnetite. The resulting black precipitated magnetite nanoparticles were later washed with excess of ethanol thrice by magnetic separation. Finally, precipitate was re-dispersed in water to obtain transparent solution of well dispersed nanoparticles. 2.3. Synthesis of amine functionalized mesoporous silica Mesoporous material was prepared by following a previously described method. Typically; 0.5 g of CTAB was first dissolved in 240 mL deionized water. Sodium hydroxide aqueous solution (2 M, 1.8 mL) was added into CTAB solution and then the reaction temperature was raised to 80 ◦ C. After stabilizing the temperature, TEOS (2.5 mL, 11.2 mmol) and 250 L APTES were consecutively added dropwise to the surfactant solution and held the reaction mixture for 2 h to give a white precipitate. The solid product was filtered, washed with deionized water and ethanol, dried at 60 ◦ C to yield the as-synthesized amine functionalized MSNPs. To maximize the anchoring of amine group, as-synthesized MSNs were refluxed for 12 h in 20 mL anhydrous toluene (300 L APTES). The product was recovered by centrifugation and washed with ethanol trice. Finally, surfactant (CTAB) was removed by refluxing the nanoparticle in acidic methanolic solution for 6 h. 2.4. Magnetite conjugation and mercapto functionalization of mesoporous silica nanoparticles Magnetite nanoparticles were anchored onto the surface of MSNs via EDC chemistry. Amine functionalized MSNs (200 mg) were dispersed in 20 mL of water. On the other hand, magnetite NPs (20 mg) were dispersed in 10 mL of water in another beaker and activated by EDC (5 mg). Both solutions were mixed to obtain magnetite conjugated MSNs (Fe3 O4 @MSNs). The solid product was then filtered, washed with deionized water and ethanol. In order to modify the remaining amine group into thiol moiety for ATO
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capturing, Fe3 O4 @MSNs nanocomposite (100 mg) was dispersed into water, and 5 mL of EDC activated MPA solution (20 L) was added into above solution. After stirring for 10 min, the product was recovered by centrifugation and washed with ethanol. 2.5. Loading of both drugs and release experiments First, ATO was immobilized onto the surface of thiol functionalized Fe3 O4 @MSNs. Thiol functionalized sample (60 mg) was dispersed into 20 mL water. Meanwhile, 50 mg of arsenic trioxide (As(OH)3 ) was dissolved in small quantity of 2 M NaOH solution and pH of the solution was adjusted to 8.0 in order to avoid silica dissolution. Equivalent amount of ATO solution was added into above Fe3 O4 @MSNs solution at room temperature. After 30 min stirring both solutions were centrifuged and washed with water. For loading hydrophobic camptothecin (CPT) in the nanochannels of mesoporous channels, ATO–Fe3 O4 @MSNs (50 mg) was introduced to DMSO solution of camptothecin (5 mL, 3 mg/mL) and stirred for 12 h at 25 ◦ C. After stirring in DMSO, the solution was transferred into 30 mL water and stirred for further 6 h. CPT was also similarly loaded in MSN nanoparticles devoid of ATO. The solution was then centrifuged and determined the amount of CPT loading by using UV/visible spectroscopy. Camptothecin and ATO release were carried out by soaking the CPT–ATO–Fe3 O4 @MSNs formulation in phosphate buffer (pH 7.4) and a dialysis bag diffusion technique was used. Briefly, 3.5 mg of samples was first dispersed in 5 mL of buffer and sealed in a dialysis bag (molecular weight cutoff = 8000). The dialysis bag was then submerged in 25 mL of the same buffer solution and stirred for five days. The released CPT in the buffer was collected at predetermined time intervals and analyzed by UV/vis spectroscopy using 365 nm wavelengths. On the other hand, ICP-OES was used to evaluate the release of arsenic. 2.6. Cell culture Pancreatic cancer cells (BxPC-3) were grown in monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Tianhang bioreagent Co., Zhejiang) and penicillin/streptomycin (100 U mL−1 and 100 g/ml, respectively) in a humidified 5% CO2 atmosphere at 37 ◦ C. 2.7. Cell viability (MTT assay) The viability of cells in the presence of nanoparticles was investigated using 3-[4,5-dimethylthialzol-2-yl]-2,5diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was carried out in triplicate in the following manner. For MTT assay, BxPC-3 were seeded into 96-well plates at a density of 8 × 103 per well in 100 L of media and grown overnight. The cells were then incubated with various Fe3 O4 @MSNs, ATOFe3 O4 @MSNs, CPT–ATO–Fe3 O4 @MSNs, and free CPT for 72 h. Afterwards, cells were incubated in media containing 0.5 mg/mL of MTT for 4 h. The precipitated formazan violet crystals were dissolved in 100 L of 10% SDS in 10 mmol HCl solution at 37 ◦ C overnight. The absorbance was measured at 570 nm by multidetection microplate reader (SynergyTM HT, BioTek Instruments Inc., USA). 2.8. Confocal laser scanning microscopy (CLSM) To check cellular uptake and CPT release, BxPC-3 were cultured in an 12-well chamber slide with one piece of cover glass at the bottom of each chamber in incubation medium (DMEM) for 4 h. CPT–ATO–Fe3 O4 @MSNs was added into the incubation medium at the concentration of 50 and 100 g/ml for 3 h incubation in 5% CO2 at 37 ◦ C. After the medium was removed, the cells were
washed twice with PBS (pH = 7.4) and the cover glass was visualized under a laser scanning confocal microscope (FluoView FV1000, Olympus). 2.9. Characterization The powder XRD patterns were recorded on a Rigaku D/Max ˚ The 2550 X-ray diffractometer with Cu-K␣ radiation ( = 1.5418 A). morphologies and detailed structure of the samples were recorded using JEOL JSM-6700F field-emission scanning electron microscope (SEM) and FEI Tecnai G2 F20 S-TWIN transmission electron micro˚ Elemental analysis was carried out on scope (TEM) ( = 1.5418 A). Perkin–Elmer ICP-OES Optima 3300DV. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer. The nitrogen adsorption and desorption isotherms were measured at liquid N2 temperature by using a Quantachrom Autosorb-iQ after the sample was degassed for 12 h at 150 ◦ C. Surface area was calculated according to the conventional BET method and the adsorption branches of the isotherms were used for the calculation of the pore parameters using the BJH method 3. Results and discussions 3.1. Characterization of multiple drug carrying theranostic nanoconstructs (ATO–CPT–Fe3 O4 @MSNs) Citric acid stabilized magnetite and MCM-41 type mesoporous silica nanoparticles were firstly prepared according to previously reported methods. The surface of mesoporous silica was functionalized with amine group via both co-condensation and post-grafting procedures to maximize the amount of amine moiety. After surfactant removal, the resulting citric acid capped magnetite nanoparticles were conjugated onto the surface of mesoporous silica particles (Fe3 O4 @MSNs) using EDC activation chemistry. In order to efficiently load ATO, the surfaces of Fe3 O4 @MSNs was functionalized with mercapto groups. Use of mercaptopropyltriethoxysilane (MPTS), as a thiol introducing agent, invariably resulted in an enhanced hydrophobicity of MSN nanoparticles, thus reducing the water dispersibility of nanocarriers. In order to avoid this restraint, mercaptopropionic acid (MPA) was used to introduce thiol functionality using EDC chemistry by reacting the amine functionalized Fe3 O4 @MSNs with carboxyl group of MPA. Finally, ATO was tethered onto the surface of Fe3 O4 @MSNs [34,35], and camptothecin (a hydrophobic topoisomerase I inhibitor) was loaded into the empty nanochannels of composite. Using UV/vis absorption measurements, loaded amount of drug was calculated to be 290 mg of CPT per gram of Fe3O4@MSNs. This exceptionally high loading of poorly water soluble CPT can be highly advantageous to improve the in vivo efficacy of this promising drug. SEM (Fig. 1a) and TEM images (Fig. 1b) of Fe3 O4 @MSNs indicates monodispersed and sphere-shaped mesoporous silica nanoparticles with a diameter of ∼100 nm. The obtained size is considered really suitable for intracellular uptake and subsequent drug release applications. The existence of 2D hexagonal porous structure with empty channels (2–3 nm) provides enough space to encapsulate relatively large amounts of therapeutic molecules. Moreover, both SEM and TEM images also verify the presence of magnetite nanocrystals, as vivid black spots onto the surface of MSNs can be clearly seen. In this study, heavy metals affinity toward thiol group was exploited for immobilizing antitumor drug ATO. It is worth mentioning that even magnetite (Fe3 O4 ) nanoparticles have previously been demonstrated as potential sorbents for arsenic removal in drinking water [36,37]. Hence, the presence of magnetite nanoparticles onto the surface of MSNs also provided an added opportunity to enhance the ATO drug loading by capitalizing
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Fig. 1. (a) SEM and (b) TEM micrographs of As–Fe3 O4 @MSNs. (c, d) High resolution TEM and HAADF-STEM survey images which indicate the existence of As, Fe, Si, and O in As–Fe3 O4 @MSNs nanoformulation.
the affinity of magnetite for heavy metals. To confirm the immobilization of ATO onto Fe3 O4 @MSNs surface, elemental analysis was carried by using energy dispersive X-ray spectroscopy (EDX) and mapping. Compositional analysis of nanocomposite was carried out
using EDX mapping (Fig. 1c and d) which confirms the presence of elemental signal of iron, arsenic and sulfur. In addition to these signals, silicon and oxygen signals (SiO2 ) are also observed in mapping. In addition to mapping, arsenic and iron signals can also be
Fig. 2. (a) Nitrogen adsorption–desorption isotherms of Fe3 O4 @MSNs. (b) EDX spectrum of As–Fe3 O4 @MSNs. (c) Wide-angle X-ray diffraction (XRD) patterns of Fe3 O4 (black) and Fe3 O4 @MSNs (red). (d) FTIR spectra of MSNs-NH2 , As–Fe3 O4 @MSNs, CPT–Fe3 O4 @MSNs and Fe3 O4 –CA. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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thiol moiety is also detected in the form of sulfur signal in the XPS analysis. The existence of SH groups in Fe3 O4 @MSNs nanocomposite is obvious from photoemission peaks of the 2p orbitals of sulfur in the SH-Fe3 O4 @MSNs spectrum at 164.0 eV (Fig. 3d), Arsenic displays an intense XPS line, As 3d, at 44.4 eV which confirms the immobilization of ATO onto Fe3 O4 @MSNs for therapeutic purposes (Fig. 3c). XPS analysis similarly provided a proof for the existence of iron with a peak at electron binding energy of 711.1 eV. 3.2. Drug release profiles of multiple drugs
Scheme 1. Schematic presentation of synthetic and loading strategies to encapsulate and tether drug cocktail molecules in magnetic porous architecture.
seen in TEM-associated EDX spectrum, suggesting the magnetite presence and conjugation of ATO in the resulting nanocomposites (Fig. 2b). Nitrogen sorption–desorption results of MSNs displayed a type IV isotherm with a hysteresis loop, indicating the existence of mesoporous structure. The mesopores size distribution indicates an intense peak placed at the mean value of 2.4 nm, implying a uniform mesoporous structure. But after magnetite conjugation, the size of hysteresis loop is decreased, as shown in Fig. 2a. Surface area (BET) of nanocomposite was also reduced to 728 from 884 m2 g−1 . Such porous architecture renders mesoporous silica as an ideal candidate for loading large quantities cytotoxicdrugs (Scheme 1). Powder X-ray diffraction was carried out to determine the purity of magnetic particles, the obtained XRD patterns (Fig. 2c) can easily be indexed to magnetite phase, whereas the magnetite conjugated MSNs nanoformulation shows similar diffraction peaks but of relatively low intensity due to the existence of silica matrix. Fourier transform infrared (FTIR) spectroscopy verified the nanoparticles conjugation and drug loading processes (Fig. 2d). Chemisorption of citric acid onto magnetite occurs through the COOH interaction. As an indication, the characteristic 1715 cm−1 peak of citric acid (C O vibration) shifts to 1618 cm−1 in citric acid passivated magnetite, suggesting the formation of complex between Fe of the magnetite surface and citric acid. This shifts or chemisorption imparts C O a partial single bond character. Moreover, a band at ∼1400 cm−1 can be assigned to the asymmetric stretching of CO from COOH moiety. Low-intensity bands between 400 and 600 cm−1 are ascribed to stretching and torsional vibration modes of magnetite. Amine functionalization of MSNs is established by the presence of bands at 3446 and 1640 cm−1 which indicates the N H stretching vibration and NH2 bending mode of free NH2 groups respectively. The appearance of bands between 2800 and 3000 cm−1 is ascribed to C H stretching vibrations of propyl group of APTES, as shown in Fig. 2d. Appearance of additional peaks in CPT loaded nanoformulation validates the successful loading of CPT. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) evaluated the quantity of arsenic in double drug nanoformulation. The concentration of arsenic was found to be 35 g per 1 mg of Fe3 O4 @MSNs. X-ray photoelectron spectroscopy (XPS) is a powerful tool to characterize the surface of a materials with a depth detection limit of about 5–10 nm, therefore, we employed this analytical technique to analyze the outermost layers of the MSNs composite to determine the elemental composition of drug loaded samples. In the survey spectrum of the ATO loaded Fe3 O4 @MSNs (Fig. 3), O, Si, Fe and arsenic signals are expectedly detected. Additionally, a small amount of carbon, nitrogen and sulfur are also detected. The C signal is basically attributed to the propyl moieties of triethoxysilane and mercaptopropionic acid. ATO capturing
In vitro release experiments were carried out to assess the release of loaded CPT from CPT–Fe3 O4 @MSN nanoformulation, under somewhat physiological conditions in PBS at pH 7.4 and at 25 ◦ C. Under these conditions, hydrophobic CPT release kinetics can be assumed to be controlled by diffusion since no appreciable degradation of mesoporous silica occurs at pH 7.4. Expectedly, the release profile (Fig. 4a) displays a controlled release pattern of anticancer drug CPT from mesoporous nanochannels which is consistent with previous reports, drug release was monitored for 5 days in this experiment [38]. In contrast to previous studies, this report achieved an exceptionally high loading of CPT (29%) in the nanochannels of porous silica. Such a high loading of hydrophobic drug in inorganic nanocarrier almost competes with various prodrug nanoformulations. Metal ions are soft acids and have strong affinity toward soft bases such as thiol groups. According to Hard/Soft Acid/Base (HSAB) theory, thiol functionalized outer surface of mesoporous silica binds with arsenic (ATO) to make a highly stable metal thiolates like sulfide minerals. Thiol arsenic bond is so stable; but a slight ATO release (10%) over the period of three days from ATO-Fe3 O4 @MSNs (Fig. 4b) points out to another important aspect which is the stability of silica nanocarrier. It is well-known that silica is slowly etched in PBS buffer (pH = 7.4), as ATO is attached onto the surface of MSNs, that is why, silica etching resulted in a slow release of chemotherapeutic agent (ATO) over a specified period of time. ICPOES results indicate that approximately 10% arsenic is untethered from the surface of MSNs. 3.3. Magnetic attributes and MRI study Besides just pinpointed delivery of drugs to diseased sites, the focus of biomedical researchers is nowadays to develop such nanoplatforms which not only ferry drugs to targeted site but also perform imaging to realize diagnosis and subsequent monitoring of therapeutic feedback. As a result, a new biomedical sub-discipline, known as theranostics, has been emerged to integrate both therapeutic and diagnostic functionalities into one nanoparticle. Magnetite nanoparticles were thus introduced to this dual drug carrying nanoparticle to serve as a theranostic agent. Magnetic hysteresis curves of both Fe3 O4 and Fe3 O4 @MSNs were acquired at room temperature using vibrating sample magnetometer, as demonstrated in Fig. 5. The magnetization curve of citric acid capped magnetite and Fe3 O4 @MSNs nanocomposite showed no hysteresis which indicates the superparamagnetic character of both nanoparticles. Saturation magnetization (Fig. 5a) of citric acid coated material was calculated to be around 80 emu g−1 . Reduction in the magnetization from 80 emu g−1 to 7 emu g−1 was observed in case of mesoporous silica-magnetite nanocomposite due to addition of higher mass fraction of silica content. Iron oxide nanocrystals are being widely utilized as contrast agents in MRI due to their negative enhancement effect on T2 -weighted sequences [39,40]. To assess the applicability of magnetite containing MSNs as MRI contrast agent, different concentrations of Fe3 O4 @MSNs were suspended in 1% agarose gel (4 mg/mL) and
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Fig. 3. (a) XPS survey spectrum of As-Fe3 O4 @MSNs, (b) Fe 2p3/2 , 2p1/2 peak, (c) As 3d and (d) S 2p.
placed in the centrifuge tubes. The MRI images of mere water and Fe3 O4 @MSNs containing tubes can be seen in Fig. 5c. T2 -weighted MR images of Fe3 O4 @MSNs samples appeared dark with increasing iron concentration due to the decrease in T2 relaxation. In contrast, tube having water remained bright and indistinguishable. MRI contrast agent performance is assessed on the basis of their
relaxivity or the alteration in the relaxation time of water protons in the presence of the agent per unit concentration. The relaxivity (r2 ) of Fe3 O4 @MSNs was found to be 161 mM−1 s−1 and can be seen in Fig. 5b. Mathematically, the r2 value of Fe3 O4 @MSNs was determined by calculating the slope of a plot of 1/T2 versus Fe concentration.
Fig. 4. (a) Release profile of camptothecin from CPT–As–Fe3 O4 @MSNs nanoformulation at pH 7.4. (b) Release profile of ATO from CPT–As–Fe3 O4 @MSNs at pH 7.4 and 25 ◦ C. The release of drug molecules was monitored by UV–vis spectrophotometer (365 nm) and inductively coupled plasma-atomic emission spectrometry (ICP-AES).
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Fig. 5. (a) M–H curves of Fe3 O4 (black line) and Fe3 O4 @MSNs (red line). Inset: photographs display water dispersibility and quick magnetophoretic separation of Fe3 O4 @MSNs nanocomposite. (b) T2 relaxation rate plots (1/T2 ) versus Fe concentration. (c) T2 -weighted MR images of dispersed Fe3 O4 @MSNs at different Fe concentrations in 1.0% agarose. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Cellular uptake, intracellular drug release and cell viability assay Confocal laser scanning microscopy (CLSM) was employed to investigate the cellular uptake and subsequent intracellular drug release from Fe3 O4 @MSN formulations (Fig. 6). Predetermined concentrations of CPT loaded Fe3 O4 @MSNs formulation was incubated with pancreatic cancer cells. Following 4 h incubation, the fluorescence micrographs clearly indicate that Fe3 O4 @MSNs nanoparticles are spontaneously internalized into BxPC-3cells. Manifestation of blue fluorescence in cytoplasmic region verified the intracellular release of CPT from ATO–CPT–Fe3 O4 @MSNs formulation as depicted in in vitro release data. Anticancer efficacy of different Fe3 O4 @MSNs formulations was tested using same pancreatic
cancer cell line (BxPC-3). Cells were treated with different concentrations of nanoformulations for 72 h, and cytotoxicity was assessed by MTT analysis. Although, the biocompatibility of magnetite and mesoporous silica is extensively reported, but we still quantified the cytotoxic potency of this composite and drugs containing Fe3 O4 @MSNs formulations. As mentioned above, arsenic and its methylated derivative are ordinarily recognized as carcinogens but now it has also been approved by FDA as an effective antileukaemic agent [41]. Extensive research has been carried out to extend the antineoplastic property of arsenic-based drugs to solid tumors. To date encouraging results about inhibiting growth and inducing apoptosis in human solid cancer cells, at clinically achievable concentrations, have been reported. Notwithstanding, its indiscriminate action and dose-dependent toxicity constrain utility of
Fig. 6. Confocal laser scanning microscopy images of BxPC-3 cells after 4 h incubation with CPT–As–Fe3 O4 @MSNs nanoformulation (50 g/ml). (a) Bright field image, (b) fluorescent image and (c) merged image. Blue fluorescence indicates the cellular uptake of and subsequent release of camptothecin. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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intracellular uptake and excellent therapeutic response in cancer cell. Exposure of ATO carrying nanoformulation effectively inhibited the growth of solid tumor cells; however, simultaneous delivery of two drugs via single nanoparticle exhibited a more pronounced cytotoxic effect probably due to drugs synergism. Merely 3 g/ml of double drug nanocarrier was found effective to exterminate tumor cells. These results advocate that the resulting magnetic nanococktail could be a promising addition in the inventory of theranostic to mitigate the severe side effects of toxic drugs on normal cells and simultaneously monitor the efficacy of chemotherapy.
Acknowledgements
Fig. 7. In vitro viability of BxPC-3 cells in the presence of different concentrations of Fe3 O4 @MSNs, As–Fe3 O4 @MSNs, CPT–Fe3 O4 @MSNs and CPT–As–Fe3 O4 @MSNs, the incubation time was 72 h.
We are grateful to the financial support from National Basic Research Program of China (973 Program, grant no. 2012CB821700), Major International (Regional) Joint Research Project of NSFC (grant no. 21120102034) and NSFC (grant no. 20831002).
References such a promising anticancer agent in solid tumors. To tackle these problems, we loaded ATO onto porous magnetic nanoparticles to specifically target cancer cells via exploiting passive and magnetic targeting approach. In vitro efficacy of ATO formulation at different concentrations was tested against BxPC-3 cells. ATO containing nanocarrier exhibited a notable killing efficiency. These results indicate that as little as 6 g/ml amount of ATO–Fe3 O4 @MSNs is enough to kill almost 50% of the cancer cells as shown in Fig. 7. As far as two drug formulations are concerned, significantly high amount of hydrophobic drug (CPT) was loaded into the channels of ATO–Fe3 O4 @MSNs, as a model drug, for ensuring efficient and controlled release of this highly promising anticancer drug, otherwise poor water solubility mars its excellent potential. This study reports a substantial amount of CPT loading (290 mg/g) compared to previously published studies, therefore, higher cell inhibition efficiency is expected in case of two drugs formulation. Anticipatedly, two drugs formulation (ATO–CPT–Fe3 O4 @MSNs) exhibits the greatest potency to inhibit the growth of BxPC-3 cell lines compared to other formulation at the same dose. Prominently, as low as 3 g/ml MSN double drug nanoformulation enabled the inhibition of almost 75% of the cancer cells. This enhanced potency can be attributed to different intracellular route for CPT; otherwise hydrophobic drug (CPT) is administered in DMSO and got precipitated in case of aqueous condition. In addition, drug synergism can also be considered a major factor for this enhanced cytotoxicity. It should also be kept in mind that two drugs cocktail not only target two different mechanisms to fight cancer but can also avoid multi drug resistance which is a major cause of chemotherapy failure due to well established mechanism to bypass resistance through endocytosis route. Based on these findings, the double drug magnetic nanoformulation can potentially be used as a platform to deliver multiple highly cytotoxic drugs specifically to cancer cells.
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4. Conclusions
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In conclusion, we have engineered a multifunctional mesoporous silica based nanocarrier to unify clinically often-prescribed combinational chemotherapy with nanotechnology. Novelty of this system lies in the exploitation of both external and internal regions of mesoporous silica to load two dissimilar cytotoxic drugs by following different approaches. In addition to drug ferrying capabilities, the resulting nanosystem also carries magnetic nanoparticles which impart magnetic manipulation and MRI diagnosis functionalities. MTT and confocal microscopy studies confirmed the
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