Journal of Colloid and Interface Science 434 (2014) 1–8
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Responsive delivery of drug cocktail via mesoporous silica nanolamps Faheem Muhammad a,b, Mingyi Guo c, Aifei Wang a, Jianyun Zhao b, Wenxiu Qi b, Yingjie Guo b, Guangshan Zhu a,d,⇑ a
State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China College of Life Science, Jilin University, Changchun 130012, PR China c College of Construction Engineering, Jilin University, Changchun 130026, PR China d Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan, QLD 4111, Australia b
a r t i c l e
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Article history: Received 30 April 2014 Accepted 18 July 2014 Available online 2 August 2014 Keywords: Controlled release Drug cocktail Image-guided therapy Mesoporous silica Quantum dots
a b s t r a c t After a substantial advancement in single drug nanocarrier, nanomedicine now demands an integration of nanotechnology with combination therapy to achieve synergistic therapeutic effects. In this respect, a smart and multiple drug shuttling nanotheranostic system is developed which transport diverse kinds of anticancer drugs to cancer cells in a controlled and responsive manner respectively. Synthetically, a significantly high dose of hydrophobic camptothecin (CPT) is first loaded into the porous structure of quantum dots (CdS) coupled mesoporous silica nanocomposite. Subsequently, fluorescent doxorubicin (DOX) molecules are exclusively anchored onto the surface of CdS; as a result, the fluorescence of both CdS and DOX is quenched. Upon exposing to mildly acidic conditions, the fluorescence of both species is recovered, such fluorescent ‘‘on–off’’ states provides an added opportunity to real time sense drug release. In-vitro cell experiment reveals an excellent anticancer efficacy of drug cocktail, merely 3 lg/ml concentration of multiple drugs loaded nanocarrier reduces the cell viability to 30%. Furthermore, confocal imaging indicates a successful release of both therapeutic entities. We visualize that our newly fabricated multifunctional double drug-carrying nanoparticles can be a valuable addition to next generation of materials that simultaneously deliver cocktail of drugs with imaging functionality. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Despite phenomenal advancement in molecular genetics, tumour biology and therapy, adequate treatment of cancer is far from satisfactory because of delayed diagnosis, indiscriminate strikes of potent cytotoxic drugs and development of chemoresistance [1,2]. Advent and integration of nanotechnology with biological systems has offered researchers a wealth of new avenues to solve this intractable health issue [3–8]. To date, variety of nanoparticle based drug delivery systems have been developed, including polymeric conjugates [9,10], micelles [11], liposomes [12], dendrimers [13], carbon nanotubes [14] and inorganic nanoparticles [15], to transport drugs in a controlled and targeted fashion [16]. While comparing drug nanocarriers, unlike liposomal and polymer-based nanoparticles, more robust and extremely stable mesoporous silica has recently emerged as one of the impressive nanotherapeutic platform [17–20]. Mesoporous silica nanoparti⇑ Corresponding author at: State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China. E-mail address: [email protected] (G. Zhu). http://dx.doi.org/10.1016/j.jcis.2014.07.024 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
cles (MSN) enjoy some exclusive properties, such as ease of synthesis, excellent biocompatibility, facile functionalization, tuneable pore structure and large surface area, which render them highly suitable for effective cell specific chemotherapy [21–24]. MSN with various functionalities have so far been successfully demonstrated to efficiently shuttle diverse kind of therapeutic agents and imaging probes into various cells, without causing any cytotoxic effects [25]. It has particularly been used to develop stimuli responsive or ‘‘smart’’ drug delivery systems which can attain site-selective delivery of anticancer drugs [26–28]. Furthermore, it can also be used to ferry multiple drugs due to the existence of both interior pores and exterior surface. Simultaneous delivery of DNA/siRNA and drugs has been demonstrated via MSN [29], however, this exclusive feature of MSN has never been used to codeliver hydrophobic and hydrophilic anticancer drugs with an aim to target two different mechanisms for enhancing the efficacy of cancer treatment. Besides drug delivery, the focus of nanomedicine is now increasingly shifted towards the development of multifunctional nanoparticles to simultaneously achieve diagnosis with therapy. When considering different imaging modalities, optical imaging is currently a widely used diagnostic tool to non-invasively detect
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disease and its progression. Quantum dots (QDs) have proficiently been applied as optical probes for real-time visualization of tumours and lymphatic system owing to their high brightness and photo-bio stability [30–32]. Few therapeutic entities have also been conjugated with quantum dots for simultaneous tumour imaging. Bagalkot et al. first developed a complex image-guided therapeutic system by conjugating targeting functionality (RNA aptamer) and doxorubicin with quantum dots (QD-Apt (DOX) [33]. The resulting system not only delivered DOX to the targeted prostate cancer cells, but also sensed the release of DOX by activating the fluorescence of the QDs presumably due to FRET phenomenon. In another report they coupled quantum dots with zwitterionic amphiphilic polymers for real-time observation of siRNA cell uptake, endosome escape, and separation from nanoparticle carrier [34]. QDs-incorporated liposomes have also been studied as nonviral drug vehicles, wherein QDs were encapsulated into the lipid bilayer of liposome to serve as fluorescence trackers [35]. Yamomoto’s group has attached captopril to the QD surface to monitor its therapeutic response and pharmacokinetics in hypertensive rats [36]. To realize a responsive combinatorial dug nanocarrier with imaging functionality, we report, for the first time, the development of luminescent CdS@MSN nanocomposite to codelivers two cytotoxic drugs simultaneously into cancer cells, in response to mildly acidic lysosomal environment (Scheme 1). 2. Materials and methods 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 (EDCHCl), tetraethyl
orthosilicate (TEOS, 99.98%), absolute ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from Aladdin reagent company. Doxorubicin hydrochloride was obtained from Yuancheng tech. development Co. Wuhan. 2.2. Synthesis of cadmium sulfide (CdS) quantum dots Following a literature approach, CdS QDs were prepared using a previous method with a slight modification [37]. Firstly, 3-mercaptopropionic acid (0.2 mmol) as stabilizing agent was introduced into 36 mL of water. After stirring for five minutes, 3 mL of Cd (NO3)2 (40 mmol) was slowly dropped into the MPA solution with constant stirring. pH of the solution was adjusted to 10–12 with tetrapropylammonium hydroxide base solution. Next, 5 mL of Na2S (20 mmol) was rapidly introduced into the system and allowed the growth of CdS nanocrystals for 10 min followed by the addition of another 5 mL of Cd (NO3)2 solution. The resulting CdS nanocrystals were separated from the solution by adding acetone. The precipitate was centrifuged and redispersed in water. 2.3. Synthesis of mesoporous silica nanosphere First of all, CTAB surfactant (1.0, 1.37 mmol) was dissolved in 240 mL of distilled water. Aqueous solution of sodium hydroxide (2.00 M, 1.75 mL) was then added to the CTAB solution and the temperature of the mixture was raised to 80 °C. Later, TEOS (2.50 mL) and APTES (250 lL) were successively added dropwise into the above surfactant solution under moderate stirring. The mixture was allowed to stir for 2 h to produce a white precipitate. The resulting solid crude product was centrifuged, washed with water and ethanol, and later dried at 60 °C. 2.4. Synthesis of amine functionalized MSN (MSN-NH2) In order to maximize the quantity of amine group, as-synthesized MSN were suspended in 50 mL of dry toluene containing 500 lL of APTES. The solution was stirred under reflux for
Scheme 1. Schematic illustration of synthetic and operational mechanism of image-guided and responsive delivery of drug cocktail.
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12 h and then product was recovered by centrifugation and washed with ethanol trice. MSN-NH2 nanoparticles were dispersed in 100 mL of ethanol containing 6 mL of HCl (37%) and reflux for 12 h to remove the pore generating template (CTAB). 2.5. Immobilization of CdS QDs, drugs loading and release procedures For loading hydrophobic camptothecin (CPT) into the nanopores of MSN, 50 mg of MSN-NH2 was introduced into DMSO solution of CPT (5 mL, 2 mg mL 1) and stirred for 12 h. DMSO solution was later added into 30 mL water and stirred the sample for further 6 h. Solution was then centrifuged and washed with PBS. In order to conjugate CdS QDs onto the surface of CPT loaded MSN-NH2 (CPTMSN), 50 mg of drug loaded sample was dispersed in 10 mL water and then 3 mg EDC coupling agent and 0.5 mL of luminescent CdS QDs (5 mg/mL) were introduced into the system and stirred the sample for 10 min. The resulting solid composite (CPT-CdS@MSN) was centrifuged, washed with water and ethanol, and later dried in air. Before loading another drug doxorubicin (DOX), carboxylic groups of CdS QDs were blocked by treating with APTES, because CdS QDs are stabilized with mercaptopropionic acid and carboxylic groups can interact with amine moiety of DOX rather than Cd 2+. CPT-CdS@MSN sample (50 mg) was added into 5 mL water, then add 50 lg APTES with 0.5 mg EDC coupling agent to form an amide bond to minimize the interaction between NH2 bearing DOX and surface bound carboxylic groups of CdS. Second drug doxorubicin was finally loaded onto CdS QDs via coordination bonding. CPTCdS@MSN (50 mg) was added into 3 mL of water containing 5 mg DOX and stirred for 30 min followed by centrifugation and washing to remove unbound drug. Loading amount of both drugs, CPT (0.267 mmol g 1) and DOX (0.055 mmol g 1),was calculated from the difference in the concentrations of the initial solution and that of the loading medium combined with the subsequent washings using UV/Vis spectroscopy at 365 and 480 nm wavelength respectively. Release kinetics of different drugs was probed by measuring in three different media (acetate buffer; pH 5.0; 6.0 and pH 7.4), using a dialysis bag diffusion technique. Briefly, 10 mg of DOX-CPT-CdS@MSN wet nanoformulations were dispersed in 5 mL of different buffer solutions, five minutes later samples were centrifuged and calculate the amount of released DOX at different pH. In case of dried powder, DOX release efficiency is relatively decreased. To determine the release behavior of CPT, which was loaded inside the nanopores, all samples were redispersed in 3 mL of PBS buffer solutions and sealed in a dialysis bag (molecular weight cutoff = 8000). The dialysis bag was in turn submerged in 20 mL of PBS solutions and stirred for 7 days. The released drug molecules in the buffer were collected at predetermined time intervals and analyzed by UV/Vis spectroscopy at 365 nm and 480 nm respectively. 2.6. Cell culture BxPc-3 (pancreatic cancer cell line) 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 lg mL 1, respectively, Gibco) in a humidified 5% CO2 atmosphere at 37 °C. 2.7. Cell viability The viability of cells in the presence of nanoparticles were investigated using 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was carried out in triplicate in the following manner. For MTT assay, BxPC-3 cells were seeded into 96-well plates at a density of 8 * 103 per well
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in 100 lL of media and grown overnight. The cells were then incubated with different concentrations of CdS@MSN, CPT-CdS@MSN and DOX-CPT-CdS@MSN for 48 h. Later on, cells were incubated in media containing 0.5 mg mL 1 of MTT for 4 h. The precipitated formazan violet crystals were dissolved in 100 lL of 10% SDS in 10 mmol HCl solution at 37 °C overnight. The absorbance was measured at 570 nm by multi-detection microplate reader (Synergy TM HT, BioTek Instruments Inc., USA).
2.8. Characterization The powder XRD patterns were recorded on a Rigaku D/Max 2550 X-ray diffractometer with Cu Ka radiation (k = 1.5418 Å). The morphologies and detailed structure of the samples were analyzed using JEOL JSM-6700F field-emission scanning electron microscope (SEM) and FEI Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) (k = 1.5418 Å). 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 120 °C. Surface area was calculated according to the conventional BET method. Fourier transform infrared (FTIR) spectra were collected on a Nicolet Impact 410 FTIR spectrometer in the range of 400–4000 cm 1. Elemental analysis was carried out on Perkin–Elmer ICP-OES Optima 3300DV. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer. PL spectra were obtained with a Perkin-Elmer LS55 luminescence spectrometer.
3. Results and discussions 3.1. Characterization of multiple drug carrying theranostic nanoconstructs (DOX-CPT-CdS@MSN) Ultrasmall, water stable CdS QDs were produced using 3-mercaptopropionic acid as stabilizing agent. Purity and crystallinity of Carboxylic terminated CdS QDs was evaluated using powder XRD investigation (Fig. S1). On the other hand, mesoporous silica nanoparticles (MSN) were prepared by following a previously established protocol and then functionalized with amine moiety (denoted as MSN-NH2) via co-condensation and post grafting approaches to achieve homogeneous and complete functionalization of amine group. Introduction of amine groups basically provides binding sites for carboxylic terminated CdS QDs and ensure the loading of DOX molecules solely to surface of CdS QDs due to repulsion between the cationic DOX and positively charged mesoporous surface. In previous studies, the release efficiency of DOX from silica nanocarrier was also quite low because of the electrostatic interaction between positively charged DOX and negatively charged silica surface. In this study, positively charged MSN ensured a significant release of DOX molecules from CdS@MSN surface in mildly acidic conditions, rather than remained bounded onto the surface of MSN due to electrostatic attraction. Electron microscopy (TEM and SEM) images revealed the average diameter (100 nm) and mesoporous structure of MSN-NH2, as clearly observed in TEM image (Fig. 1b and c) The N2 adsorption/desorption analysis of MSN-NH2 indicated the mesoporous structure with a typical type IV isotherm. The BET surface area of MSNs was found to be 840 m2/g (Fig. S2). The porous structure and suitable particle size of MSN can be better used as a nanoplatform for the fabrication of responsive combinatorial drug delivery system. X-ray diffraction (XRD) pattern, as shown in Fig. S3, indicates a typical ordered mesoporous structure of MSN. To render MSN luminescent, carboxylic terminated CdS QDs were then conjugated onto the surface of MSN. TEM investigation of CdS@MSN nanocomposite
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Fig. 1. (a) TEM image of CdS QDs, (b) SEM micrograph of CdS conjugated MSN, (c) TEM micrograph of CdS tethered MSN and (d) HAADF-STEM survey images indicating the existence of Cd, Si, S and O in CdS@MSN nanocomposite.
showed an obvious presence of 3–4 nm QDs, as clearly noticeable dark spots on the surface of the MSN can be seen in Fig. 1c. Moreover, the compositional analysis of nanocomposite was carried out using EDX mapping (Fig. 1d), which verified the presence of elemental signal of the both cadmium and sulphur. In addition to those signals, silicon and oxygen signals, which demonstrate silica, are also observed in EDX analysis. Similarly, X-ray photoelectron spectra (XPS) proved the conjugation of QDs in MSN drug formulation. In the survey spectrum of the CdS@MSN (Fig. 2), mainly O, Si, and Cd signals are found, as were expected. In addition, a small amount of carbon, nitrogen and sulphur was also detected. The C signal was mainly attributed to the propyl groups of triethoxysilane and mercaptopropionic acid. High resolution spectrum contained Cd 3d specific peaks (405.25 eV and 412.5 eV), moreover, the quantification of peaks of Cd 3d and S 2p provided the ratio of Cd/S as 60:40 (Fig. 2b and c). The presence of slightly more divalent cadmium ions (Cd2+) provide conjugating sites for loading DOX. Following CdS immobilization, the surface area of MSN was slightly decreased from 840 to 730 m2/g, due to partial blockage of nanopores by CdS QDs (Fig. S2). Nevertheless, the surface area of CdS@MSN was sufficiently large to be used for loading and release of a hydrophobic drug in the nanochannels. Finally, luminescent doxorubicin (DOX) molecules were anchored onto the surface of luminescent QDs, resulting in a quenching of both luminescent species. We have previously reported that the incubation of DOX with divalent metal ions (Zn2+) leads to the formation of a reversible complex [38]. The stability of the resulting drug-metal complex mainly depends upon pH of solution [39,40]. In this study Cd2+ chelated with DOX molecules at physiological pH, however, that bond was readily cleaved in mildly acidic conditions due to intense competition between metal ions and proton (H+) to conjugate with Lewis base (DOX). To probe the complexation of DOX with CdS, absorption and fluorescence spectroscopy were employed. Furthermore; chelate formation can also be detected by naked eye. The red shift from 479 to 539 nm in the typical spectrum of DOX suggested the
deprotonation of DOX at phenolate groups (Fig. 3a). Shift in the main band of the DOX towards slightly higher energy is also indicating the involvement of carbonyl group of DOX in metal complexation. A colour change in DOX solution was from orange to pink observed after the addition of CdS QDs (inset of Fig. 3d). While considering the luminescent properties of both CdS and DOX, fluorescence spectroscopy offered an invaluable assistance to validate their interaction at neutral pH. Successive reduction in the fluorescence intensity of QDs was observed, when a fixed amount of CdS QDs (100 lg) were incubated with an increasing amount of DOX (10–70 lg) (Fig. 3b). Likewise, with increasing CdS QDs concentration (20–100 lg), a corresponding decrease in the native fluorescence spectrum of DOX (50 lg) was observed which finally led to a maximum level of fluorescence quenching, as it can be seen in Fig. 3c. These results imply a coordination mediated binding between DOX and divalent metal ions. As it is mentioned above that complexation process heavily relies on pH of the solution, therefore, coordinate bond can easily cleaved by changing the pH. The pH sensitive behaviour was also authenticated using fluorescence spectroscopy through adjusting the pH of DOX-CdS@MSN solution. Fig. 3d demonstrates an instant recovery of DOX fluorescence when the pH value of DOX-CdS@MSN formulation was lowered by adding acetate buffer (pH 5.0). Most interestingly, the luminescence CdS QDs was also recovered after DOX release in physiological pH. Acid-induced fluorescence recovery of DOX can be ascribed to the dissociation of the Cd2+-DOX bond. The inset of Fig. 3d displays a photograph of pink of DOX-CdS@MSN sample in water (pH 7.0), whereas the cleavage of bond and subsequent recovery of DOX in acetate buffer (5.0) is shown in the inset of Fig. 3d (orange colour). Fluorescent on–off states can be highly valuable to sense the delivery of anticancer drugs and also track the therapeutic efficacy of chemotherapy. The surface modifications and drug loading was validated via FTIR spectra (Fig. 2d). The chemisorption of mercaptopropionic acid (MPA) onto the surface of CdS QDs was shown by appearance of a prominent peak
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Fig. 2. (a) Survey spectrum of XPS Spectra of CdS@ MSN, (b) Cd 3d, (c) S 2p, (d) FTIR spectra of MSN-NH2, CPT-MSN, CPT-CdS@MSN, DOX-CPT-CdS@MSN and CdS-MPA QDs.
at 1710 cm 1 due to the C@O stretch for the protonated carboxylic acid. Another band at 3030 cm 1 is ascribed to OH stretching of ACOOH group and the CAH stretching of alkyl groups. Amine functionalization of MSN was verified through band at 1640 cm 1, which indicated the NH2 bending mode of free NH2 groups. After loading camptothecin into MSN-NH2, characteristics CPT bands 1750 cm 1 1602 cm 1 were emerged, which were not present in drug-free nanoparticles. The DOX loading onto the surface of CPT-CdS@MSN formulation was supported by the existence of band at 1585 cm 1 which was designated to the stretching vibration of two carbonyl groups of the anthracene ring of DOX.
positively charged NH2 group, a fair quantity of DOX is bound onto MSNs surface and subsequent release quantity is greatly suppressed due to the electrostatic interaction between negatively charged silanol groups and cationic DOX. This system accomplished a considerable DOX release from nanocomposite. It is worth noting that this nanocomposite possesses both kinds of release profiles, hydrophobic CPT is sustainably released from mesoporous channels, whereas DOX follows a burst-like release property after reaching its targeted site (acidic condition). Owing to the existence of lower pH values (as low as 5.7) in cancer cells than normal cells (pH 7.4), acid responsive anticancer drug release can be an effective targeted chemotherapeutic strategy.
3.2. Responsive drug release profiles of multiple drugs 3.3. Cellular uptake, intracellular drug release and Cell viability assay Fig. 4a presents in vitro drug release profiles of two drugs from DOX-CPT-CdS@MSN nanoformulation in PBS pH 7.4, and in acetate buffers (6.0, 5.0). Release data demonstrated a sustained release pattern of hydrophobic CPT from nanochannels of MSN. Notably, a negligible amount of DOX was discharged after 7 days at pH 7.4, however, exposure of double drug formulation to mildly acidic environ (pH 6–5) yielded a burst-like release pattern of DOX (70%) mainly due to the cleavage of metal-DOX chelate (Fig. 4b). As mentioned above, surface functionalization is of significant importance in altering the release kinetics; amine functionalization of MSNs in this study results in the detachment of considerable amount of DOX from MSN surface. In the absence of
To investigate the in vitro chemotherapeutic efficacy, the cytotoxicity of different CdS@MSN formulations against pancreatic cancer cells (Bxpc-3) was evaluated by MTT cell viability assay. Different nanoformulations (in a concentration range of 0–200 lg/ mL) were incubated with pancreatic cancer cells for 48 h. The biocompatibility of mesoporous silica is well established, however, the cell viability data of CdS@MSN reveals a slight cytotoxicity, which most probably stems from the presence of cadmium (Fig. S4). Notably, the quantity of CdS was quite low (5% doping), that is why such a minute amount of cadmium cannot compete with two extremely lethal anticancer drugs. Camptothecin (CPT)
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Fig. 3. (a) Absorbance spectra of free DOX (orange) and DOX-CdS QDs (pink) in aqueous solutions. (b) Fluorescence spectra of CdS QDs with increasing DOX concentration from 2.5 to 22.5 lg (top to bottom). The excitation wavelength is 350 nm and the pH is 7.4. (c) Fluorescence spectra of DOX with increasing concentration of CdS QDs from 5 to 50 lL (top to bottom). The excitation wavelength is 480 nm and the pH is 7.4. (d) Fluorescence recovery of DOX at pH 5.0: Inset photograph displays. (a) Under white light, CdS-MPA QDs in water. (b) Under UV light, CdS-MPA QDs in water. (c) Under white light, DOX-CdS@MSN at pH 7.4. (d) Under UV light, DOX-CdS@MSN at pH 7.4. (e) Under white light, DOX-CdS@MSN at pH 5.0. (f) Under UV light, DOX-CdS@MSN at pH 5.0. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. (a) Release profile of camptothecin from DOX-CPT-CdS@MSN nanoformulation at pH 7.4. (b) Release profile of doxorubicin from DOX-CPT-CdS@MSN at pH 7.4, 6.0 and 5.0. The release of drug molecules was monitored by UV–Vis spectrophotometer.
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Fig. 5. In vitro viability of BxPC-3 cells in the presence of different concentrations of CPT-CdS@MSN, DOX-CdS@MSN and DOX-CPT-CdS@MSN, the incubation time was 48 h.
Fig. 6. CLSM micrographs of BxPC-3 cells after 4 h incubation with DOX-CPT-CdS@MSN (50 lg/mL) (a, e, i) transmission image of cancer cells. (b) Blue fluorescence indicates the release of CPT from nanoporoes of silica. (f) Green fluorescence implies the recovery of CdS luminesense after the release of DOX. (j) Intense red fluorescence indicates the pH responsive release of quenched DOX, (c, g, k) overlaid images of bright and fluorescent images, (d, h, l) fluorescent images obtained by merging two and/or three luminescent species. For instance, image d is obtained by merging all three fluorescent images (b, f, j). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
is one of the most promising anticancer drugs against several carcinomas in vitro, but its hydrophobic nature hampers its effectiveness in vivo. To tackle this problem, a significant dose (93 mg/g) of hydrophobic CPT was encapsulated into the nanochannels of CdS@MSN. In comparison to mere CdS@MSN nanocomposite, killing efficacy of CPT formulation was found to be exceptionally high
against cancer cells due to high loading and controlled drug release. Even a really low concentration (6 lg/mL) of CPTCdS@MSN is enough to attain 70% cell reduction, whereas the corresponding concentration of CPT free CdS@MSN formulation was unable to cause any cell damage, proving the biocompatibility of CdS containing nanocomposite (Fig. 5). For the sake of comparison,
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DOX loaded CdS@MSN formulation was also evaluated, which also proved to be highly potent and yields a concentration-dependent significant decrease in cell count with IC50 (half-maximal inhibitory concentration) of (12 lg/mL). Two drugs formulation expectedly exhibitedthe greatest potency to inhibit the growth of Bxpc-3 cell lines compared to one drug formulations. IC50 of DOX-CPT-CdS@MSN against cells was calculated to be less than 3 lg/mL, relatively high therapeutic effect of DOX-CPT-CdS@MSN indicates an effective intracellular release of DOX in response to acidifying lysosomal environment. Confocal laser scanning microscopy was employed to monitor cellular uptake, sense intracellular DOX release and image the cancer cells. Pancreatic cancer cell line (Bxpc-3) was incubated with 100 lg/mL of various nanoformulations of CdS@MSN for different periods of time at 37 °C. Incubation was followed by copious washing with phosphate-buffered saline (PBS) solution to remove unbound nanoparticles. Cellular uptake of CdS@MSN formulation was observed, as CdS radiated strong green fluorescence under UV excitation, indicated the intracellular uptake of nanocarrier (Fig. S5). To verify the release of both drugs and sense the DOX transport, Bxpc-3 cells were similarly incubated with 100 lg/mL of fluorescence ‘‘OFF’’ double drug ferrying nanocomposite sample (DOX-CPT-CdS@MSN). After four hours incubation, cell images indicated green dots and red and blue spots in cell bodies, suggesting the efficient uptake and subsequent release of both drugs from the nanoformulation. Blue spots in Fig. 6a–c implied the release of CPT molecules from nanopores of mesoporous silica, whereas the intense red (Fig. 6i–k) and green spots (Fig. 6e–g) respectively signified the release of DOX and luminescence recovery of CdS QDs. Detachment of conjugated DOX molecules from the quantum dots, after intracellular pH stimulation, resulted in the recovery of fluorescence of both species. Cell micrographs (d, h, l) in Fig. 6 illustrates merged fluorescence of all three luminescent components. Thus, this acid sensitive On–off state offers an opportunity to monitor the drug release and later therapeutic response of the therapy.
Acknowledgments 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) NSFC (Grant No. 20831002) and Australian Research Council Future Fellowship (FT100101059). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.07.024. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
4. Conclusions In conclusion, a smart and multifunctional nanoconstruct was developed to merge combinational therapy with nanotechnology. Porous structure of silica was employed to load a lethal dose of hydrophobic drug in nanopores, whereas CdS QDs studded external surface was exploited to load doxorubicin (DOX) exclusively onto CdS surface via coordination chemistry. Notably, the fluorescence of both QDs and DOX was turned ‘‘OFF’’ after DOX loading, however, exposure to mildly acidic conditions prompted the fluorescence of QDs and DOX to ‘‘ON’’ state due to the cleavage of coordinate bond between DOX and QDs. pH dependent fluorescent ‘‘on–off’’ states provided an exceptional opportunity to not only deliver anticancer drugs, but also sense their release. Drug cocktail exhibited an outstanding cell inhibiting efficiency and the confocal imaging established a successful delivery of both anticancer drugs to BxPc-3 cells. We envision that our newly fabricated multifunctional double drug-carrying nanoparticles can be a valuable addition to next generation of materials that simultaneously deliver cocktail of drugs and sense their intracellular release in real time.
[23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
J.S. Bertram, Mol. Aspects Med. 21 (2000) 167. M.A. Moses, H. Brem, R. Langer, Cancer Cell 4 (2003) 337. R. Duncan, R. Gaspar, Mol. Pharm. 8 (2011) 2101. M.E. Davis, Z. Chen, D.M. Shin, Nat. Rev. Drug Discov. 7 (2008) 771. T. Kubik, K. Bogunia-Kubik, M. Sugisaka, Curr. Pharm. Biotechnol. 6 (2005) 17. M.C. Roco, Curr. Opin. Biotechnol. 14 (2003) 337. G.M. Whitesides, Nat. Biotech. 21 (2003) 1161. K. Cho, X. Wang, S. Nie, Z. Chen, D.M. Shin, Clin. Cancer Res. 14 (2008) 1310. R. Duncan, Nat. Rev. Drug Discov. 2 (2003) 347. R. Duncan, Nat. Rev. Cancer 6 (2006) 688. K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 47 (2001) 113. C. Kirby, G. Gregoriadis, Nat. Biotechnol. 2 (1984) 979. E.R. Gillies, J.M.J. Fréchet, Drug Discovery Today 10 (2005) 35. Z. Liu, K. Chen, C. Davis, S. Sherlock, Q. Cao, X. Chen, H. Dai, Cancer Res. 68 (2008) 6652. S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R.N. Muller, Chem. Rev. 108 (2008) 2064. O.C. Farokhzad, J. Cheng, B.A. Teply, I. Sherifi, S. Jon, P.W. Kantoff, J.P. Richie, R. Langer, Proc. Natl. Acad. Sci. 103 (2006) 6315. M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 46 (2007) 7548. S.-H. Wu, Y. Hung, C.-Y. Mou, Chem. Commun. 47 (2011) 9972. I.I. Slowing, B.G. Trewyn, S. Giri, V.S.Y. Lin, Adv. Funct. Mater. 17 (2007) 1225. J. Liu, A. Stace-Naughton, X. Jiang, C.J. Brinker, J. Am. Chem. Soc. 131 (2009) 1354. J.E. Lee, N. Lee, H. Kim, J. Kim, S.H. Choi, J.H. Kim, T. Kim, I.C. Song, S.P. Park, W.K. Moon, T. Hyeon, J. Am. Chem. Soc. 132 (2009) 552. C.E. Ashley, E.C. Carnes, G.K. Phillips, D. Padilla, P.N. Durfee, P.A. Brown, T.N. Hanna, J. Liu, B. Phillips, M.B. Carter, N.J. Carroll, X. Jiang, D.R. Dunphy, C.L. Willman, D.N. Petsev, D.G. Evans, A.N. Parikh, B. Chackerian, W. Wharton, D.S. Peabody, C.J. Brinker, Nat. Mater. 10 (2011) 389. X. Du, B. Shi, J. Liang, J. Bi, S. Dai, S.Z. Qiao, Adv. Mater. 25 (2013) 5981. X. Du, B. Shi, Y. Tang, S. Dai, S.Z. Qiao, Biomaterials 35 (2014) 5580. J.M. Rosenholm, E. Peuhu, J.E. Eriksson, C. Sahlgren, M. Linde´n, Nano Lett. 9 (2009) 3308. R. Liu, Y. Zhang, X. Zhao, A. Agarwal, L.J. Mueller, P. Feng, J. Am. Chem. Soc. 132 (2010) 1500. F. Muhammad, M. Guo, W. Qi, F. Sun, A. Wang, Y. Guo, G. Zhu, J. Am. Chem. Soc. 133 (2011) 8778. A. Popat, B.P. Ross, J. Liu, S. Jambhrunkar, F. Kleitz, S.Z. Qiao, Angew. Chem. Int. Ed. 51 (2012) 12486. H. Meng, M. Liong, T. Xia, Z. Li, Z. Ji, J.I. Zink, A.E. Nel, ACS Nano 4 (2010) 4539. I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435. T.M. Jovin, Nat. Biotechnol. 21 (2003) 32. X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Science 307 (2005) 538. V. Bagalkot, L. Zhang, E. Levy-Nissenbaum, S. Jon, P.W. Kantoff, R. Langer, O.C. Farokhzad, Nano Lett. 7 (2007) 3065. M.V. Yezhelyev, L. Qi, R.M. O’Regan, S. Nie, X. Gao, J. Am. Chem. Soc. 130 (2008) 9006. L. Qi, X. Gao, ACS Nano 2 (2008) 1403. N. Manabe, A. Hoshino, L. Yi-qiang, T. Goto, N. Kato, S. Yamamoto, NanoBiosci., IEEE Trans. 5 (2006) 263. M. Xue, X. Wang, H. Wang, B. Tang, Talanta 83 (2011) 1680. F. Muhammad, M. Guo, Y. Guo, W. Qi, F. Qu, F. Sun, H. Zhao, G. Zhu, J. Mater. Chem. 21 (2011) 13406. M.M.L. Fiallo, A. Garnier-Suillerot, Biochemistry 25 (1986) 924. M.M.L. Fiallo, A. Garnier-Suillerot, B. Matzanke, H. Kozlowski, J. Inorg. Biochem. 75 (1999) 105.
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Journal of Colloid and Interface Science www.elsevier.com/locate/jcis
Responsive delivery of drug cocktail via mesoporous silica nanolamps Faheem Muhammad a,b, Mingyi Guo c, Aifei Wang a, Jianyun Zhao b, Wenxiu Qi b, Yingjie Guo b, Guangshan Zhu a,d,⇑ a
State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China College of Life Science, Jilin University, Changchun 130012, PR China c College of Construction Engineering, Jilin University, Changchun 130026, PR China d Queensland Micro- and Nanotechnology Centre, Griffith University, Nathan, QLD 4111, Australia b
a r t i c l e
i n f o
Article history: Received 30 April 2014 Accepted 18 July 2014 Available online 2 August 2014 Keywords: Controlled release Drug cocktail Image-guided therapy Mesoporous silica Quantum dots
a b s t r a c t After a substantial advancement in single drug nanocarrier, nanomedicine now demands an integration of nanotechnology with combination therapy to achieve synergistic therapeutic effects. In this respect, a smart and multiple drug shuttling nanotheranostic system is developed which transport diverse kinds of anticancer drugs to cancer cells in a controlled and responsive manner respectively. Synthetically, a significantly high dose of hydrophobic camptothecin (CPT) is first loaded into the porous structure of quantum dots (CdS) coupled mesoporous silica nanocomposite. Subsequently, fluorescent doxorubicin (DOX) molecules are exclusively anchored onto the surface of CdS; as a result, the fluorescence of both CdS and DOX is quenched. Upon exposing to mildly acidic conditions, the fluorescence of both species is recovered, such fluorescent ‘‘on–off’’ states provides an added opportunity to real time sense drug release. In-vitro cell experiment reveals an excellent anticancer efficacy of drug cocktail, merely 3 lg/ml concentration of multiple drugs loaded nanocarrier reduces the cell viability to 30%. Furthermore, confocal imaging indicates a successful release of both therapeutic entities. We visualize that our newly fabricated multifunctional double drug-carrying nanoparticles can be a valuable addition to next generation of materials that simultaneously deliver cocktail of drugs with imaging functionality. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Despite phenomenal advancement in molecular genetics, tumour biology and therapy, adequate treatment of cancer is far from satisfactory because of delayed diagnosis, indiscriminate strikes of potent cytotoxic drugs and development of chemoresistance [1,2]. Advent and integration of nanotechnology with biological systems has offered researchers a wealth of new avenues to solve this intractable health issue [3–8]. To date, variety of nanoparticle based drug delivery systems have been developed, including polymeric conjugates [9,10], micelles [11], liposomes [12], dendrimers [13], carbon nanotubes [14] and inorganic nanoparticles [15], to transport drugs in a controlled and targeted fashion [16]. While comparing drug nanocarriers, unlike liposomal and polymer-based nanoparticles, more robust and extremely stable mesoporous silica has recently emerged as one of the impressive nanotherapeutic platform [17–20]. Mesoporous silica nanoparti⇑ Corresponding author at: State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, PR China. E-mail address: [email protected] (G. Zhu). http://dx.doi.org/10.1016/j.jcis.2014.07.024 0021-9797/Ó 2014 Elsevier Inc. All rights reserved.
cles (MSN) enjoy some exclusive properties, such as ease of synthesis, excellent biocompatibility, facile functionalization, tuneable pore structure and large surface area, which render them highly suitable for effective cell specific chemotherapy [21–24]. MSN with various functionalities have so far been successfully demonstrated to efficiently shuttle diverse kind of therapeutic agents and imaging probes into various cells, without causing any cytotoxic effects [25]. It has particularly been used to develop stimuli responsive or ‘‘smart’’ drug delivery systems which can attain site-selective delivery of anticancer drugs [26–28]. Furthermore, it can also be used to ferry multiple drugs due to the existence of both interior pores and exterior surface. Simultaneous delivery of DNA/siRNA and drugs has been demonstrated via MSN [29], however, this exclusive feature of MSN has never been used to codeliver hydrophobic and hydrophilic anticancer drugs with an aim to target two different mechanisms for enhancing the efficacy of cancer treatment. Besides drug delivery, the focus of nanomedicine is now increasingly shifted towards the development of multifunctional nanoparticles to simultaneously achieve diagnosis with therapy. When considering different imaging modalities, optical imaging is currently a widely used diagnostic tool to non-invasively detect
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disease and its progression. Quantum dots (QDs) have proficiently been applied as optical probes for real-time visualization of tumours and lymphatic system owing to their high brightness and photo-bio stability [30–32]. Few therapeutic entities have also been conjugated with quantum dots for simultaneous tumour imaging. Bagalkot et al. first developed a complex image-guided therapeutic system by conjugating targeting functionality (RNA aptamer) and doxorubicin with quantum dots (QD-Apt (DOX) [33]. The resulting system not only delivered DOX to the targeted prostate cancer cells, but also sensed the release of DOX by activating the fluorescence of the QDs presumably due to FRET phenomenon. In another report they coupled quantum dots with zwitterionic amphiphilic polymers for real-time observation of siRNA cell uptake, endosome escape, and separation from nanoparticle carrier [34]. QDs-incorporated liposomes have also been studied as nonviral drug vehicles, wherein QDs were encapsulated into the lipid bilayer of liposome to serve as fluorescence trackers [35]. Yamomoto’s group has attached captopril to the QD surface to monitor its therapeutic response and pharmacokinetics in hypertensive rats [36]. To realize a responsive combinatorial dug nanocarrier with imaging functionality, we report, for the first time, the development of luminescent CdS@MSN nanocomposite to codelivers two cytotoxic drugs simultaneously into cancer cells, in response to mildly acidic lysosomal environment (Scheme 1). 2. Materials and methods 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 (EDCHCl), tetraethyl
orthosilicate (TEOS, 99.98%), absolute ethanol, dimethyl sulfoxide (DMSO) and toluene were obtained from Aladdin reagent company. Doxorubicin hydrochloride was obtained from Yuancheng tech. development Co. Wuhan. 2.2. Synthesis of cadmium sulfide (CdS) quantum dots Following a literature approach, CdS QDs were prepared using a previous method with a slight modification [37]. Firstly, 3-mercaptopropionic acid (0.2 mmol) as stabilizing agent was introduced into 36 mL of water. After stirring for five minutes, 3 mL of Cd (NO3)2 (40 mmol) was slowly dropped into the MPA solution with constant stirring. pH of the solution was adjusted to 10–12 with tetrapropylammonium hydroxide base solution. Next, 5 mL of Na2S (20 mmol) was rapidly introduced into the system and allowed the growth of CdS nanocrystals for 10 min followed by the addition of another 5 mL of Cd (NO3)2 solution. The resulting CdS nanocrystals were separated from the solution by adding acetone. The precipitate was centrifuged and redispersed in water. 2.3. Synthesis of mesoporous silica nanosphere First of all, CTAB surfactant (1.0, 1.37 mmol) was dissolved in 240 mL of distilled water. Aqueous solution of sodium hydroxide (2.00 M, 1.75 mL) was then added to the CTAB solution and the temperature of the mixture was raised to 80 °C. Later, TEOS (2.50 mL) and APTES (250 lL) were successively added dropwise into the above surfactant solution under moderate stirring. The mixture was allowed to stir for 2 h to produce a white precipitate. The resulting solid crude product was centrifuged, washed with water and ethanol, and later dried at 60 °C. 2.4. Synthesis of amine functionalized MSN (MSN-NH2) In order to maximize the quantity of amine group, as-synthesized MSN were suspended in 50 mL of dry toluene containing 500 lL of APTES. The solution was stirred under reflux for
Scheme 1. Schematic illustration of synthetic and operational mechanism of image-guided and responsive delivery of drug cocktail.
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12 h and then product was recovered by centrifugation and washed with ethanol trice. MSN-NH2 nanoparticles were dispersed in 100 mL of ethanol containing 6 mL of HCl (37%) and reflux for 12 h to remove the pore generating template (CTAB). 2.5. Immobilization of CdS QDs, drugs loading and release procedures For loading hydrophobic camptothecin (CPT) into the nanopores of MSN, 50 mg of MSN-NH2 was introduced into DMSO solution of CPT (5 mL, 2 mg mL 1) and stirred for 12 h. DMSO solution was later added into 30 mL water and stirred the sample for further 6 h. Solution was then centrifuged and washed with PBS. In order to conjugate CdS QDs onto the surface of CPT loaded MSN-NH2 (CPTMSN), 50 mg of drug loaded sample was dispersed in 10 mL water and then 3 mg EDC coupling agent and 0.5 mL of luminescent CdS QDs (5 mg/mL) were introduced into the system and stirred the sample for 10 min. The resulting solid composite (CPT-CdS@MSN) was centrifuged, washed with water and ethanol, and later dried in air. Before loading another drug doxorubicin (DOX), carboxylic groups of CdS QDs were blocked by treating with APTES, because CdS QDs are stabilized with mercaptopropionic acid and carboxylic groups can interact with amine moiety of DOX rather than Cd 2+. CPT-CdS@MSN sample (50 mg) was added into 5 mL water, then add 50 lg APTES with 0.5 mg EDC coupling agent to form an amide bond to minimize the interaction between NH2 bearing DOX and surface bound carboxylic groups of CdS. Second drug doxorubicin was finally loaded onto CdS QDs via coordination bonding. CPTCdS@MSN (50 mg) was added into 3 mL of water containing 5 mg DOX and stirred for 30 min followed by centrifugation and washing to remove unbound drug. Loading amount of both drugs, CPT (0.267 mmol g 1) and DOX (0.055 mmol g 1),was calculated from the difference in the concentrations of the initial solution and that of the loading medium combined with the subsequent washings using UV/Vis spectroscopy at 365 and 480 nm wavelength respectively. Release kinetics of different drugs was probed by measuring in three different media (acetate buffer; pH 5.0; 6.0 and pH 7.4), using a dialysis bag diffusion technique. Briefly, 10 mg of DOX-CPT-CdS@MSN wet nanoformulations were dispersed in 5 mL of different buffer solutions, five minutes later samples were centrifuged and calculate the amount of released DOX at different pH. In case of dried powder, DOX release efficiency is relatively decreased. To determine the release behavior of CPT, which was loaded inside the nanopores, all samples were redispersed in 3 mL of PBS buffer solutions and sealed in a dialysis bag (molecular weight cutoff = 8000). The dialysis bag was in turn submerged in 20 mL of PBS solutions and stirred for 7 days. The released drug molecules in the buffer were collected at predetermined time intervals and analyzed by UV/Vis spectroscopy at 365 nm and 480 nm respectively. 2.6. Cell culture BxPc-3 (pancreatic cancer cell line) 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 lg mL 1, respectively, Gibco) in a humidified 5% CO2 atmosphere at 37 °C. 2.7. Cell viability The viability of cells in the presence of nanoparticles were investigated using 3-[4,5-dimethylthialzol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. The assay was carried out in triplicate in the following manner. For MTT assay, BxPC-3 cells were seeded into 96-well plates at a density of 8 * 103 per well
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in 100 lL of media and grown overnight. The cells were then incubated with different concentrations of CdS@MSN, CPT-CdS@MSN and DOX-CPT-CdS@MSN for 48 h. Later on, cells were incubated in media containing 0.5 mg mL 1 of MTT for 4 h. The precipitated formazan violet crystals were dissolved in 100 lL of 10% SDS in 10 mmol HCl solution at 37 °C overnight. The absorbance was measured at 570 nm by multi-detection microplate reader (Synergy TM HT, BioTek Instruments Inc., USA).
2.8. Characterization The powder XRD patterns were recorded on a Rigaku D/Max 2550 X-ray diffractometer with Cu Ka radiation (k = 1.5418 Å). The morphologies and detailed structure of the samples were analyzed using JEOL JSM-6700F field-emission scanning electron microscope (SEM) and FEI Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) (k = 1.5418 Å). 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 120 °C. Surface area was calculated according to the conventional BET method. Fourier transform infrared (FTIR) spectra were collected on a Nicolet Impact 410 FTIR spectrometer in the range of 400–4000 cm 1. Elemental analysis was carried out on Perkin–Elmer ICP-OES Optima 3300DV. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer. PL spectra were obtained with a Perkin-Elmer LS55 luminescence spectrometer.
3. Results and discussions 3.1. Characterization of multiple drug carrying theranostic nanoconstructs (DOX-CPT-CdS@MSN) Ultrasmall, water stable CdS QDs were produced using 3-mercaptopropionic acid as stabilizing agent. Purity and crystallinity of Carboxylic terminated CdS QDs was evaluated using powder XRD investigation (Fig. S1). On the other hand, mesoporous silica nanoparticles (MSN) were prepared by following a previously established protocol and then functionalized with amine moiety (denoted as MSN-NH2) via co-condensation and post grafting approaches to achieve homogeneous and complete functionalization of amine group. Introduction of amine groups basically provides binding sites for carboxylic terminated CdS QDs and ensure the loading of DOX molecules solely to surface of CdS QDs due to repulsion between the cationic DOX and positively charged mesoporous surface. In previous studies, the release efficiency of DOX from silica nanocarrier was also quite low because of the electrostatic interaction between positively charged DOX and negatively charged silica surface. In this study, positively charged MSN ensured a significant release of DOX molecules from CdS@MSN surface in mildly acidic conditions, rather than remained bounded onto the surface of MSN due to electrostatic attraction. Electron microscopy (TEM and SEM) images revealed the average diameter (100 nm) and mesoporous structure of MSN-NH2, as clearly observed in TEM image (Fig. 1b and c) The N2 adsorption/desorption analysis of MSN-NH2 indicated the mesoporous structure with a typical type IV isotherm. The BET surface area of MSNs was found to be 840 m2/g (Fig. S2). The porous structure and suitable particle size of MSN can be better used as a nanoplatform for the fabrication of responsive combinatorial drug delivery system. X-ray diffraction (XRD) pattern, as shown in Fig. S3, indicates a typical ordered mesoporous structure of MSN. To render MSN luminescent, carboxylic terminated CdS QDs were then conjugated onto the surface of MSN. TEM investigation of CdS@MSN nanocomposite
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Fig. 1. (a) TEM image of CdS QDs, (b) SEM micrograph of CdS conjugated MSN, (c) TEM micrograph of CdS tethered MSN and (d) HAADF-STEM survey images indicating the existence of Cd, Si, S and O in CdS@MSN nanocomposite.
showed an obvious presence of 3–4 nm QDs, as clearly noticeable dark spots on the surface of the MSN can be seen in Fig. 1c. Moreover, the compositional analysis of nanocomposite was carried out using EDX mapping (Fig. 1d), which verified the presence of elemental signal of the both cadmium and sulphur. In addition to those signals, silicon and oxygen signals, which demonstrate silica, are also observed in EDX analysis. Similarly, X-ray photoelectron spectra (XPS) proved the conjugation of QDs in MSN drug formulation. In the survey spectrum of the CdS@MSN (Fig. 2), mainly O, Si, and Cd signals are found, as were expected. In addition, a small amount of carbon, nitrogen and sulphur was also detected. The C signal was mainly attributed to the propyl groups of triethoxysilane and mercaptopropionic acid. High resolution spectrum contained Cd 3d specific peaks (405.25 eV and 412.5 eV), moreover, the quantification of peaks of Cd 3d and S 2p provided the ratio of Cd/S as 60:40 (Fig. 2b and c). The presence of slightly more divalent cadmium ions (Cd2+) provide conjugating sites for loading DOX. Following CdS immobilization, the surface area of MSN was slightly decreased from 840 to 730 m2/g, due to partial blockage of nanopores by CdS QDs (Fig. S2). Nevertheless, the surface area of CdS@MSN was sufficiently large to be used for loading and release of a hydrophobic drug in the nanochannels. Finally, luminescent doxorubicin (DOX) molecules were anchored onto the surface of luminescent QDs, resulting in a quenching of both luminescent species. We have previously reported that the incubation of DOX with divalent metal ions (Zn2+) leads to the formation of a reversible complex [38]. The stability of the resulting drug-metal complex mainly depends upon pH of solution [39,40]. In this study Cd2+ chelated with DOX molecules at physiological pH, however, that bond was readily cleaved in mildly acidic conditions due to intense competition between metal ions and proton (H+) to conjugate with Lewis base (DOX). To probe the complexation of DOX with CdS, absorption and fluorescence spectroscopy were employed. Furthermore; chelate formation can also be detected by naked eye. The red shift from 479 to 539 nm in the typical spectrum of DOX suggested the
deprotonation of DOX at phenolate groups (Fig. 3a). Shift in the main band of the DOX towards slightly higher energy is also indicating the involvement of carbonyl group of DOX in metal complexation. A colour change in DOX solution was from orange to pink observed after the addition of CdS QDs (inset of Fig. 3d). While considering the luminescent properties of both CdS and DOX, fluorescence spectroscopy offered an invaluable assistance to validate their interaction at neutral pH. Successive reduction in the fluorescence intensity of QDs was observed, when a fixed amount of CdS QDs (100 lg) were incubated with an increasing amount of DOX (10–70 lg) (Fig. 3b). Likewise, with increasing CdS QDs concentration (20–100 lg), a corresponding decrease in the native fluorescence spectrum of DOX (50 lg) was observed which finally led to a maximum level of fluorescence quenching, as it can be seen in Fig. 3c. These results imply a coordination mediated binding between DOX and divalent metal ions. As it is mentioned above that complexation process heavily relies on pH of the solution, therefore, coordinate bond can easily cleaved by changing the pH. The pH sensitive behaviour was also authenticated using fluorescence spectroscopy through adjusting the pH of DOX-CdS@MSN solution. Fig. 3d demonstrates an instant recovery of DOX fluorescence when the pH value of DOX-CdS@MSN formulation was lowered by adding acetate buffer (pH 5.0). Most interestingly, the luminescence CdS QDs was also recovered after DOX release in physiological pH. Acid-induced fluorescence recovery of DOX can be ascribed to the dissociation of the Cd2+-DOX bond. The inset of Fig. 3d displays a photograph of pink of DOX-CdS@MSN sample in water (pH 7.0), whereas the cleavage of bond and subsequent recovery of DOX in acetate buffer (5.0) is shown in the inset of Fig. 3d (orange colour). Fluorescent on–off states can be highly valuable to sense the delivery of anticancer drugs and also track the therapeutic efficacy of chemotherapy. The surface modifications and drug loading was validated via FTIR spectra (Fig. 2d). The chemisorption of mercaptopropionic acid (MPA) onto the surface of CdS QDs was shown by appearance of a prominent peak
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Fig. 2. (a) Survey spectrum of XPS Spectra of CdS@ MSN, (b) Cd 3d, (c) S 2p, (d) FTIR spectra of MSN-NH2, CPT-MSN, CPT-CdS@MSN, DOX-CPT-CdS@MSN and CdS-MPA QDs.
at 1710 cm 1 due to the C@O stretch for the protonated carboxylic acid. Another band at 3030 cm 1 is ascribed to OH stretching of ACOOH group and the CAH stretching of alkyl groups. Amine functionalization of MSN was verified through band at 1640 cm 1, which indicated the NH2 bending mode of free NH2 groups. After loading camptothecin into MSN-NH2, characteristics CPT bands 1750 cm 1 1602 cm 1 were emerged, which were not present in drug-free nanoparticles. The DOX loading onto the surface of CPT-CdS@MSN formulation was supported by the existence of band at 1585 cm 1 which was designated to the stretching vibration of two carbonyl groups of the anthracene ring of DOX.
positively charged NH2 group, a fair quantity of DOX is bound onto MSNs surface and subsequent release quantity is greatly suppressed due to the electrostatic interaction between negatively charged silanol groups and cationic DOX. This system accomplished a considerable DOX release from nanocomposite. It is worth noting that this nanocomposite possesses both kinds of release profiles, hydrophobic CPT is sustainably released from mesoporous channels, whereas DOX follows a burst-like release property after reaching its targeted site (acidic condition). Owing to the existence of lower pH values (as low as 5.7) in cancer cells than normal cells (pH 7.4), acid responsive anticancer drug release can be an effective targeted chemotherapeutic strategy.
3.2. Responsive drug release profiles of multiple drugs 3.3. Cellular uptake, intracellular drug release and Cell viability assay Fig. 4a presents in vitro drug release profiles of two drugs from DOX-CPT-CdS@MSN nanoformulation in PBS pH 7.4, and in acetate buffers (6.0, 5.0). Release data demonstrated a sustained release pattern of hydrophobic CPT from nanochannels of MSN. Notably, a negligible amount of DOX was discharged after 7 days at pH 7.4, however, exposure of double drug formulation to mildly acidic environ (pH 6–5) yielded a burst-like release pattern of DOX (70%) mainly due to the cleavage of metal-DOX chelate (Fig. 4b). As mentioned above, surface functionalization is of significant importance in altering the release kinetics; amine functionalization of MSNs in this study results in the detachment of considerable amount of DOX from MSN surface. In the absence of
To investigate the in vitro chemotherapeutic efficacy, the cytotoxicity of different CdS@MSN formulations against pancreatic cancer cells (Bxpc-3) was evaluated by MTT cell viability assay. Different nanoformulations (in a concentration range of 0–200 lg/ mL) were incubated with pancreatic cancer cells for 48 h. The biocompatibility of mesoporous silica is well established, however, the cell viability data of CdS@MSN reveals a slight cytotoxicity, which most probably stems from the presence of cadmium (Fig. S4). Notably, the quantity of CdS was quite low (5% doping), that is why such a minute amount of cadmium cannot compete with two extremely lethal anticancer drugs. Camptothecin (CPT)
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Fig. 3. (a) Absorbance spectra of free DOX (orange) and DOX-CdS QDs (pink) in aqueous solutions. (b) Fluorescence spectra of CdS QDs with increasing DOX concentration from 2.5 to 22.5 lg (top to bottom). The excitation wavelength is 350 nm and the pH is 7.4. (c) Fluorescence spectra of DOX with increasing concentration of CdS QDs from 5 to 50 lL (top to bottom). The excitation wavelength is 480 nm and the pH is 7.4. (d) Fluorescence recovery of DOX at pH 5.0: Inset photograph displays. (a) Under white light, CdS-MPA QDs in water. (b) Under UV light, CdS-MPA QDs in water. (c) Under white light, DOX-CdS@MSN at pH 7.4. (d) Under UV light, DOX-CdS@MSN at pH 7.4. (e) Under white light, DOX-CdS@MSN at pH 5.0. (f) Under UV light, DOX-CdS@MSN at pH 5.0. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. (a) Release profile of camptothecin from DOX-CPT-CdS@MSN nanoformulation at pH 7.4. (b) Release profile of doxorubicin from DOX-CPT-CdS@MSN at pH 7.4, 6.0 and 5.0. The release of drug molecules was monitored by UV–Vis spectrophotometer.
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Fig. 5. In vitro viability of BxPC-3 cells in the presence of different concentrations of CPT-CdS@MSN, DOX-CdS@MSN and DOX-CPT-CdS@MSN, the incubation time was 48 h.
Fig. 6. CLSM micrographs of BxPC-3 cells after 4 h incubation with DOX-CPT-CdS@MSN (50 lg/mL) (a, e, i) transmission image of cancer cells. (b) Blue fluorescence indicates the release of CPT from nanoporoes of silica. (f) Green fluorescence implies the recovery of CdS luminesense after the release of DOX. (j) Intense red fluorescence indicates the pH responsive release of quenched DOX, (c, g, k) overlaid images of bright and fluorescent images, (d, h, l) fluorescent images obtained by merging two and/or three luminescent species. For instance, image d is obtained by merging all three fluorescent images (b, f, j). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
is one of the most promising anticancer drugs against several carcinomas in vitro, but its hydrophobic nature hampers its effectiveness in vivo. To tackle this problem, a significant dose (93 mg/g) of hydrophobic CPT was encapsulated into the nanochannels of CdS@MSN. In comparison to mere CdS@MSN nanocomposite, killing efficacy of CPT formulation was found to be exceptionally high
against cancer cells due to high loading and controlled drug release. Even a really low concentration (6 lg/mL) of CPTCdS@MSN is enough to attain 70% cell reduction, whereas the corresponding concentration of CPT free CdS@MSN formulation was unable to cause any cell damage, proving the biocompatibility of CdS containing nanocomposite (Fig. 5). For the sake of comparison,
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DOX loaded CdS@MSN formulation was also evaluated, which also proved to be highly potent and yields a concentration-dependent significant decrease in cell count with IC50 (half-maximal inhibitory concentration) of (12 lg/mL). Two drugs formulation expectedly exhibitedthe greatest potency to inhibit the growth of Bxpc-3 cell lines compared to one drug formulations. IC50 of DOX-CPT-CdS@MSN against cells was calculated to be less than 3 lg/mL, relatively high therapeutic effect of DOX-CPT-CdS@MSN indicates an effective intracellular release of DOX in response to acidifying lysosomal environment. Confocal laser scanning microscopy was employed to monitor cellular uptake, sense intracellular DOX release and image the cancer cells. Pancreatic cancer cell line (Bxpc-3) was incubated with 100 lg/mL of various nanoformulations of CdS@MSN for different periods of time at 37 °C. Incubation was followed by copious washing with phosphate-buffered saline (PBS) solution to remove unbound nanoparticles. Cellular uptake of CdS@MSN formulation was observed, as CdS radiated strong green fluorescence under UV excitation, indicated the intracellular uptake of nanocarrier (Fig. S5). To verify the release of both drugs and sense the DOX transport, Bxpc-3 cells were similarly incubated with 100 lg/mL of fluorescence ‘‘OFF’’ double drug ferrying nanocomposite sample (DOX-CPT-CdS@MSN). After four hours incubation, cell images indicated green dots and red and blue spots in cell bodies, suggesting the efficient uptake and subsequent release of both drugs from the nanoformulation. Blue spots in Fig. 6a–c implied the release of CPT molecules from nanopores of mesoporous silica, whereas the intense red (Fig. 6i–k) and green spots (Fig. 6e–g) respectively signified the release of DOX and luminescence recovery of CdS QDs. Detachment of conjugated DOX molecules from the quantum dots, after intracellular pH stimulation, resulted in the recovery of fluorescence of both species. Cell micrographs (d, h, l) in Fig. 6 illustrates merged fluorescence of all three luminescent components. Thus, this acid sensitive On–off state offers an opportunity to monitor the drug release and later therapeutic response of the therapy.
Acknowledgments 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) NSFC (Grant No. 20831002) and Australian Research Council Future Fellowship (FT100101059). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcis.2014.07.024. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
4. Conclusions In conclusion, a smart and multifunctional nanoconstruct was developed to merge combinational therapy with nanotechnology. Porous structure of silica was employed to load a lethal dose of hydrophobic drug in nanopores, whereas CdS QDs studded external surface was exploited to load doxorubicin (DOX) exclusively onto CdS surface via coordination chemistry. Notably, the fluorescence of both QDs and DOX was turned ‘‘OFF’’ after DOX loading, however, exposure to mildly acidic conditions prompted the fluorescence of QDs and DOX to ‘‘ON’’ state due to the cleavage of coordinate bond between DOX and QDs. pH dependent fluorescent ‘‘on–off’’ states provided an exceptional opportunity to not only deliver anticancer drugs, but also sense their release. Drug cocktail exhibited an outstanding cell inhibiting efficiency and the confocal imaging established a successful delivery of both anticancer drugs to BxPc-3 cells. We envision that our newly fabricated multifunctional double drug-carrying nanoparticles can be a valuable addition to next generation of materials that simultaneously deliver cocktail of drugs and sense their intracellular release in real time.
[23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]
J.S. Bertram, Mol. Aspects Med. 21 (2000) 167. M.A. Moses, H. Brem, R. Langer, Cancer Cell 4 (2003) 337. R. Duncan, R. Gaspar, Mol. Pharm. 8 (2011) 2101. M.E. Davis, Z. Chen, D.M. Shin, Nat. Rev. Drug Discov. 7 (2008) 771. T. Kubik, K. Bogunia-Kubik, M. Sugisaka, Curr. Pharm. Biotechnol. 6 (2005) 17. M.C. Roco, Curr. Opin. Biotechnol. 14 (2003) 337. G.M. Whitesides, Nat. Biotech. 21 (2003) 1161. K. Cho, X. Wang, S. Nie, Z. Chen, D.M. Shin, Clin. Cancer Res. 14 (2008) 1310. R. Duncan, Nat. Rev. Drug Discov. 2 (2003) 347. R. Duncan, Nat. Rev. Cancer 6 (2006) 688. K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 47 (2001) 113. C. Kirby, G. Gregoriadis, Nat. Biotechnol. 2 (1984) 979. E.R. Gillies, J.M.J. Fréchet, Drug Discovery Today 10 (2005) 35. Z. Liu, K. Chen, C. Davis, S. Sherlock, Q. Cao, X. Chen, H. Dai, Cancer Res. 68 (2008) 6652. S. Laurent, D. Forge, M. Port, A. Roch, C. Robic, L. Vander Elst, R.N. Muller, Chem. Rev. 108 (2008) 2064. O.C. Farokhzad, J. Cheng, B.A. Teply, I. Sherifi, S. Jon, P.W. Kantoff, J.P. Richie, R. Langer, Proc. Natl. Acad. Sci. 103 (2006) 6315. M. Vallet-Regí, F. Balas, D. Arcos, Angew. Chem. Int. Ed. 46 (2007) 7548. S.-H. Wu, Y. Hung, C.-Y. Mou, Chem. Commun. 47 (2011) 9972. I.I. Slowing, B.G. Trewyn, S. Giri, V.S.Y. Lin, Adv. Funct. Mater. 17 (2007) 1225. J. Liu, A. Stace-Naughton, X. Jiang, C.J. Brinker, J. Am. Chem. Soc. 131 (2009) 1354. J.E. Lee, N. Lee, H. Kim, J. Kim, S.H. Choi, J.H. Kim, T. Kim, I.C. Song, S.P. Park, W.K. Moon, T. Hyeon, J. Am. Chem. Soc. 132 (2009) 552. C.E. Ashley, E.C. Carnes, G.K. Phillips, D. Padilla, P.N. Durfee, P.A. Brown, T.N. Hanna, J. Liu, B. Phillips, M.B. Carter, N.J. Carroll, X. Jiang, D.R. Dunphy, C.L. Willman, D.N. Petsev, D.G. Evans, A.N. Parikh, B. Chackerian, W. Wharton, D.S. Peabody, C.J. Brinker, Nat. Mater. 10 (2011) 389. X. Du, B. Shi, J. Liang, J. Bi, S. Dai, S.Z. Qiao, Adv. Mater. 25 (2013) 5981. X. Du, B. Shi, Y. Tang, S. Dai, S.Z. Qiao, Biomaterials 35 (2014) 5580. J.M. Rosenholm, E. Peuhu, J.E. Eriksson, C. Sahlgren, M. Linde´n, Nano Lett. 9 (2009) 3308. R. Liu, Y. Zhang, X. Zhao, A. Agarwal, L.J. Mueller, P. Feng, J. Am. Chem. Soc. 132 (2010) 1500. F. Muhammad, M. Guo, W. Qi, F. Sun, A. Wang, Y. Guo, G. Zhu, J. Am. Chem. Soc. 133 (2011) 8778. A. Popat, B.P. Ross, J. Liu, S. Jambhrunkar, F. Kleitz, S.Z. Qiao, Angew. Chem. Int. Ed. 51 (2012) 12486. H. Meng, M. Liong, T. Xia, Z. Li, Z. Ji, J.I. Zink, A.E. Nel, ACS Nano 4 (2010) 4539. I.L. Medintz, H.T. Uyeda, E.R. Goldman, H. Mattoussi, Nat. Mater. 4 (2005) 435. T.M. Jovin, Nat. Biotechnol. 21 (2003) 32. X. Michalet, F.F. Pinaud, L.A. Bentolila, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Science 307 (2005) 538. V. Bagalkot, L. Zhang, E. Levy-Nissenbaum, S. Jon, P.W. Kantoff, R. Langer, O.C. Farokhzad, Nano Lett. 7 (2007) 3065. M.V. Yezhelyev, L. Qi, R.M. O’Regan, S. Nie, X. Gao, J. Am. Chem. Soc. 130 (2008) 9006. L. Qi, X. Gao, ACS Nano 2 (2008) 1403. N. Manabe, A. Hoshino, L. Yi-qiang, T. Goto, N. Kato, S. Yamamoto, NanoBiosci., IEEE Trans. 5 (2006) 263. M. Xue, X. Wang, H. Wang, B. Tang, Talanta 83 (2011) 1680. F. Muhammad, M. Guo, Y. Guo, W. Qi, F. Qu, F. Sun, H. Zhao, G. Zhu, J. Mater. Chem. 21 (2011) 13406. M.M.L. Fiallo, A. Garnier-Suillerot, Biochemistry 25 (1986) 924. M.M.L. Fiallo, A. Garnier-Suillerot, B. Matzanke, H. Kozlowski, J. Inorg. Biochem. 75 (1999) 105.
