A Versatile Theranostic Delivery Platform Integrating Magnetic

May 10, 2016 - Tracking embryonic hematopoietic stem cells to the bone marrow: nanoparticle options to evaluate transplantation efficiency. Sean K. Sw...
0 downloads 0 Views 3MB Size
Download PDF
A Versatile Theranostic Delivery Platform Integrating Magnetic Resonance Imaging/ Computed Tomography, pH/cis-Diol Controlled Release, and Targeted Therapy Yu-Jui Tseng,† Shang-Wei Chou,† Jing-Jong Shyue,#,⊥ Shih-Yao Lin,† Jong-Kai Hsiao,*,‡,§ and Pi-Tai Chou*,† †

Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan Department of Medical Imaging, Taipei TzuChi Hospital, The Buddhist Tzuchi Medical Foundation, Taipei 23142, Taiwan # Department of Materials Science and Engineering, National Taiwan University, Taipei 10617, Taiwan ⊥ Research Center for Applied Science, Academia Sinica, Taipei 11529, Taiwan § School of Medicine, Tzu-Chi University, Hualien 97004, Taiwan ‡

S Supporting Information *

ABSTRACT: The functions of biomedical imaging, cancer targeting, and controlled release of therapeutic agents were integrated into a drug delivery platform to proof its diagnostic and therapeutic capabilities. This versatile nanocomposite is based on the strategic design of wormlike mesoporous silica nanocarriers that are decorated with extremely small iron oxide nanoparticles, having a prominent T1-weighted Magnetic Resonance Imaging (MRI) signal. The controlled release function was then achieved through the grafting of polyalcohol saccharide derivative ligands onto the surfaces of mesoporous silica nanoparticles to conjugate with boronic acid functionalized gold nanoparticles, which acted as the gate and the source of computed tomography (CT) signals. This versatile platform thus exhibited a MRI/CT dual imaging property drawing on the strong points to offset the weaknesses of each, rendering more accurate diagnosis. The capping of gold nanoparticles controlled with the hydrolysis of boronate ester bonds provides the reversible opening/closing process, avoiding further release of drug once the nanocomposite leaves the cell or tissue. To endow this platform with targeting ability, protocatechuic acid was utilized as a linker to connect folic acid with the boronic acid of the gold nanoparticles. The anchor of targeting moiety, folic acid, enriched this platform and enhanced the specific cellular uptake toward cells with folate receptor. This integrated drug delivery platform was then loaded with the antitumor agent doxorubicin, demonstrating its power for targeted delivery, bioimaging, and controlled release chemotherapy to reduce the undesired side effects of chemotherapy. KEYWORDS: T1-weighted iron oxide nanoparticles, dual imaging, reversible controlled release, targeted therapy, theranostic platform

F

the delivery site because of a lack of proper protective screens. These disadvantages can be overcome by encapsulating the drug delivery system with gatekeepers to prevent leakage. This idea is the basis of a stimuli-responsive controlled release drug delivery system, in which payloads are stored and protected in the systems and their release is triggered when they arrive at the delivery site.

ollowing the disclosure of human genome, individualized medicine combining targeted imaging and therapy toward neoplasm is in great demand. However, the combined treatment agent was not possible until the development of theranostic nanomedicine.1−3 The delivery of therapeutic agents within nanoparticles presents challenges in a physiological environment, so the equipment for imaging, targeting, and other functions in the delivery systems must be improved.4,5 Due to the high surface area of the nanostructure, mesoporous silica nanoparticles are currently the favorable choice of delivery vehicle.6−9 However, with a traditional delivery system, some leakage inevitably occurs on the path to © 2016 American Chemical Society

Received: December 23, 2015 Accepted: May 10, 2016 Published: May 10, 2016 5809

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

www.acsnano.org

Article

ACS Nano

proliferation of neural stem cell and preventing their apoptotic cell death, so it is a suitable agent for the treatment of Parkinson’s disease.36 PCA then links to graft folic acid (FA) that has high affinity to folate receptors (FRs), which can be found overexpressed and anchored on cancer cell membranes. As elaborated in the following sections, our multifunctional drug delivery system synergistically combines features of both diagnostic and therapeutic capabilities.

Magnetic resonance imaging (MRI) is a common imaging modality in both clinical and biological applications due to its zero ionizing radiation, unlimited tissue penetration, and noninvasive nature.10,11 Traditional T1-weighted contrast agents are Gd-based12−16 and Mn-based17,18 complexes or nanoparticles, which are hazardous once they dissociate and accumulate in the body. On the other hand, contrast agents such as superparamagnetic iron oxides (SPIOs) are well-known for the high intensity of their T2-weighted signals.19−21 Unlike T1-weighted contrast agents, T2 signals, which produce negative contrast, may cause confusion in recognizing normal tissues. Recently, extremely small iron oxide nanoparticles (ESIONPs) with suppressed magnetization have attracted attention for their potential as T1 contrast agents because they have a good T1 contrast effect and are biocompatible due to the capability of the human body to metabolize iron ions, a natural component of hemoglobin.22 The conjugation of nanoparticles with targeting moiety that recognizes receptors overexpressed on the surface of a tumor cell will prevent nonspecific uptake by normal cells and increase the possibility of transporting particles to an intended destination.23,24 Many researchers are currently studying nanoparticles and molecules that can be exploited as nanogates for mesoporous silica nanocarriers,25−33 but few have endowed their controlled release drug delivery systems with targeting ability, particularly not those with reversible gates. The difficulties are mainly due to impediments such as a lack of space for grafting targeting ligands and the necessity of controlling the reaction conditions when grafting targeting ligands onto the surface of capped drug delivery systems.34,35 The triggering methods of controlled release drug delivery systems usually depend on stimuli such as pH, temperature or specific additives, but the drug stored inside the system is usually also pH- or thermo-sensitive. Since unnecessary changes are unwanted during the functionalization of targeting molecules, reaction environments should be mild. Furthermore, if targeting ligands are tethered onto the capping agents or the carrier surface first, they could affect the capping of the nanogates. In this study, we present the design and synthesis of a multifunctional drug delivery nanoplatform with three main functions: (1) T1-weighted MRI/CT dual imaging capability, (2) pH/cis-diol dual-responsive reversible controlled release drug delivery system, and (3) tumor targeting of folate receptors (FRs) with folic acid (FA). We utilize wormlike mesoporous silica nanoparticles (WMSNs) as the core, on which extremely small iron oxide nanoparticles (ESIONPs) are adsorbed on the surface. ESIONPs on the WMSN are then encapsulated in a thin mesoporous silica shell. The incorporation of ESIONPs with WMSN gives the system a T1-weighted MRI signal. To endow the system with controlled release functionality, we tether polyalcohol saccharide-derivative silane on the surface of the system to bind with boronic acid functionalized gold nanoparticles (BAuNPs) via the formation of boronate ester bonds, which are a reversible reaction and can be hydrolyzed or condensed by changing pH or in the presence of cis-diol. Moreover, the capping of gold nanoparticles provides an extra CT imaging function. To demonstrate the possibility of targeted therapy, we use 3,4-dihydroxybenzoic acid (protocatechuic acid, PCA), which is known for its antioxidative properties, suppressing cancer growth in some cell types such as human hepatocellular carcinoma as our linker. PCA is also capable of promoting the

RESULTS AND DISCUSSION Synthesis and Characterization of the As-Synthesized Materials. The extremely small iron oxide nanoparticles (ESIONPs) were synthesized following the reported literature with some modifications. Figure S1 shows a transmission electron microscopy (TEM) image of the dispersed, uniform ESIONPs with an average diameter of ca. 2 nm. In a typical synthesis protocol, maghemite nanoparticles were prepared using a thermal decomposition reaction with iron−oleate complex as the precursor, oleyl alcohol as the reductant, and 1octadecane as the solvent. When the reaction mixture was aged at 200 °C, 2 nm ESIONPs of uniform size were produced. The 2 nm-sized nanoparticles had a maghemite crystal structure. The ESIONPS were highly stable in organic solvents such as chloroform or hexane. We recently reported a T1 MRI contrast agent that grew FeOOH inside the channel of wormlike mesoporous silica nanoparticle.37 Here, we demonstrate another method to encapsulate ESIONPs within mesoporous silica. To combine ESIONPs with wormhole-like mesoporous silica nanoparticles (WMSNs), we first synthesized WMSNs with an average diameter of ∼50 nm. Note that MCM-41 is often used as a drug carrier because of its unique architecture featuring parallel pores with two unique openings. However, the difficulty of encapsulating multifunctional nanoparticles inside the network of MCM-41 makes further modification difficult. We then synthesized wormhole-like mesoporous silica nanoparticles, which had four domains: core, channel, network, and outer surface,38−40 to be used as our drug delivery system basis. For the synthesis of ESIONPs@WMSNs nanocomposites, the asprepared ESIONPs with hydrophobic ligands capped and dispersed in chloroform were transferred to H2O by surface modification with CTAB surfactants. For CTAB surfactant coating, van der Waals interactions between the hydrophobic chains and CTAB led to water-soluble magnetic nanoparticles with hydrophilic positive charged head groups of CTAB outward. Subsequently, the WMSNs were poured into the ESIONPS solution. The electrostatic interaction between the positive surface charges of the ESIONPs and negative surface charges of WMSNs caused the ESIONPs to adsorb onto the surface of the WMSNs. The resulted nanoparticles were further accessed using hydrolysis condensation of the TEOS molecules to form a mesoporous silica shell around the ESIONPs adsorbed WMSNs and thus successfully encapsulate the ESIONPs into the network of mesoporous silica nanoparticles. The TEOS condensation reaction was done with NH4F added. CTAB micelle is the organic template that interacts with the silicate polyanion product from the base-catalyzed hydrolysis of TEOS. This allowed the assembly of silicate polyanion and CTAB surfactants to form a mesoporous silica shell on the nanoparticle surface. The necessity of the WMSN core can be shown by performing the same protocol without the core. As shown by the results in Figure S2, the nanoparticles form severe aggregates, indicating the importance of the WMSN core. 5810

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Scheme 1. Structure of FA-PCA-BAuNPs-Ligand-mS-ESIONPs@WMSN and Illustration of Targeted Drug Delivery in a Cell

dihydroxybenzoic acid as the linker, which possesses two hydroxylic groups with the ortho position and formed ester bonds with the phenylboronic acid on the surface of the gold nanoparticles. This bond can also be hydrolyzed by changes in pH and cis-diols. Then, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and n-hydroxysuccinimide (EDC/ NHS) combined with carboxylic acid of 3,4-dihydroxybenzoic acid and the amino group of folic acid were used to form an amide bond. For clarity, the overall reaction pathway and illustration of targeted drug delivery in a cell are depicted in Scheme 1. Figure 1a presents a transmission electron micrograph of the wormhole-like mesoporous silica nanoparticles (WMSNs), showing that they had an average size of ∼50 nm, a narrow size distribution and good dispersity. Figure S3 shows the ESIONPs adsorbed on the surface by the electrostatic interaction of the negative-charged surfaces of the WMSNs and the positive-charged of the hydrophilic CTAB headgroup of ESIONPs’ ligands. A large number of ESIONPs were on the surface of the WMSNs, as indicated by the appearance of tiny gray dots in Figure S3. After the ESIONPs@WMSNs were encapsulated by the addition of tetraethyl orthosilicate (TEOS), there was no obvious difference between the assynthesized mS-ESIONPs@WMSNs and WMSNs. This could be attributed to the extremely small of the ESIONPs, which lowered the degree of discrimination owing to the screen of the

Without the WMSN core, the ESIONPs would not be adsorbed, causing the random sol−gel polymerization of TEOS onto the CTAB micelle. The distinctive characteristics of functionalized mesoporous silica supports, such as high homogeneous porosity, inertness, thermal stability, tunability of homogeneous pore sizes, and the feasibility of functionalizing external or internal surfaces, make such scaffold ideal for hosting functional guest molecules. The surface anchored mS-ESIONP@WMSN nanocomposites with N-(3-triethoxysilylpropyl)gluconamide can be used as a controllable release drug delivery system due to the formation of boronate ester bonds, which is a reversible reaction between saccharide derivative ligands and phenylboronic acid functionalized gold nanoparticles. Boronate esters can be hydrolyzed under acidic conditions or in the presence of cis-diols.41−43 When the saccharide derivative ligands interacted with the boronic acid, the gold nanoparticles cap onto the surface of WMSNs and act as nanoscopic gates. As a well stimuli triggered controllable gating system, the boronate ester based gating system can be triggered by two simple external stimuli, either pH changes or the addition of cis-diols such as glucose or mannitol. The extra advantage of boronic acid capped nanoscopic gating is the possibility for further modification of targeting molecules such as RGD peptide or folic acid with the help of boronate ester bond formation. Here we applied 3,45811

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 1. TEM images of (a) WMSNs, (b) mS-ESIONPs@WMSNs, (c) BAuNPs, (d) FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSNs, (e) UV−vis spectrum of folic acid, BAuNPs-ligand-mS-ESIONPs@WMSNs and FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSNs.

addition, folic acid absorption bands at 284 and 360 nm after the conjugation of folic acid were observed. Since sulfur has better sensitivity and was distributed on the surface of BAuNPs, we then used its auger electron signal via the Scanning Auger Microscope (SAM) measurement to map the image. The results shown in Figure 2a reveal the strong sulfur signal on the surface of BAuNPs-ligand-mS-ESIONPs@WMSN (distributed as spheres), providing additional support of the capping of BAuNPs. Evidence is also given by the good overlap between SAM (Figure 2a) and its corresponding SEM (Figure 2b). Moreover, mapping the corresponding EELS (electron energy loss spectrometry) has been performed to show the composition of the as-prepared nanocomposite (Figure 2c− h). The results with pseudocolor clearly reveal the distribution of Si (blue), Au (yellow), and Fe (red) elements in each individual nanostructure. Also, the HRTEM images show lattice fringes corresponding to a spacing distance of 1.48 Å, which is the (440) facet of γ-Fe2O3 (Figure S5c,d) and 2.36 Å, which is the (111) facet of gold nanoparticles (Figure S5d), providing further evidence for the functionalization of ESIONPs and BAuNPs. The adsorption of ESIONPs on the WMSNs (cores) following the encapsulation of ESIONPs@WMSNs in a mesoporous silica shell and the capping of B-AuNPs were further verified with X-ray Photoelectron Spectroscopy (XPS). In Figure S6, the binding energy of ESIONPs at about 710 and 725 eV can be attributed to the binding energy of Fe 2p3/2 and Fe 2p1/2. Given that the detection depth of XPS was 5 nm, after the encapsulation of ESIONPs, the signals of Fe 2p3/2 and Fe 2p1/2 are shown in Figure 3a, which ensures that the ESIONPs

mesoporous silica shell. The graft of the saccharide derivative ligands, N-(3-triethoxysilylpropyl)gluconamide utilized the silanol group of N-(3-triethoxysilylpropyl)gluconamide to form covalent bonds via hydrolysis and condensation reaction with the silanol groups of the mesoporous silica and tether to the silica surface (Figure 1b). The boronic acid functionalized nanoparticles (B-AuNPs) were then applied to cap the pores of the WMSNs through the conjugation of the boronic acid and the N-(3-triethoxysilylpropyl)gluconamide. The average size of the B-AuNPs was about 3.5 nm (Figure 1c), and they were remarkably homogeneous. The rate of formation of the boronate ester bond was high once the B-AuNPs were added to the solution of ligand-mS-ESIONPs@WMSNs. It is obvious in Figure 1d that B-AuNPs (dark contrast) covered the entire surface of the ligand-mS-ESIONPs@WMSNs, showing that BAuNPs indeed acted as the nanogate and capped the pores. The immobilization of 3, 4-dihydroxybenzoic acid and the targeting molecules did not greatly change the outward appearance (Figure 1d). Figure 1e shows the UV−visible spectrum of FA-PCABAuNPs-mS-ESIONPs@WMSNs and BAuNPs-mSESIONPs@WMSNs. The local maximum at 530 nm of the B-AuNPs spectrum is ascribed to the surface plasmon resonance band (Figure S4). Therefore, the local maximum at ∼550 nm for BAuNPs-mS-ESIONPs@WMSNs ensures the capping of B-AuNPs onto the mS-ESIONPs@WMSNs. It should be mentioned that the slight difference in the local maxima of these two samples might be due to the slight aggregation of BAuNPs resulting from the crowded arrangement on the surface of the ligand-mS-ESIONPs@WMSNs. In 5812

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 2. (a) Scanning auger microscopy (SAM) of BAuNPs-ligand-mS-ESIONPs@WMSNs. (b) The corresponding SEM of panel a. Element mapping by electron energy loss spectrometry (EELS); (c) Si, (d) Fe, (e) Au, (f) merged image of Au and Si, (g) merged image of Fe, Au, and Si. (h) ZLP (zero-loss peak) image of BAuNPs-ligand-mS-ESIONPs@WMSN.

surface area was lost due to the conjugation of particles and ligands, appreciable porosity was still available for drug loading. BJH (Barrett−Joyner−Halenda) analysis (Figure S9b) showed pore size distributions, in which the WMSNs, mS-ESIONPs@ WMSNs, and ligand-mS-ESIONPs@WMSNs nanoparticles had similar pore size distribution at about 2 nm, the range of mesopore. After BAuNPs were capped, the specific surface area fell to 131 m2/g and the pore size distribution decreased to a diameter smaller than about 1.5 nm, which is out of the range of data collection points, indicating efficient blocking of the pores (Figure S9a,b). We also performed small-angle X-ray diffraction to gather evidence of the mesoporous structure of the WMSNs. The ordering of mesoporous of WMSNs appeared at 2θ near 2.2°, which is the diffraction intensity of the (100) peak of WMSNs (Figure S10). Wide angle X-ray diffraction (XRD) was used to unveil the iron-relevant diffraction peaks from mS-ESIONPs@WMSNs. Despite the

existed in the shallow layer of mS-ESIONPs@WMSNs instead of in the deep core, where the signal of ESIONPs might not be detected. The full XPS spectrum of mS-ESIONPs@WMSN and the corresponding detailed spectra of major component elements are shown in Figure S7. The block of the Fe 2p3/2 and Fe 2p1/2 could be seen as well after the capping of B-AuNPs. The ∼3.5 nm-sized B-AuNPs were conjugated onto the surface of ligand-mS-ESIONPs@WMSNs, increasing the relative depth of the ESIONPs and finally decreasing the Fe 2p3/2 and Fe 2p1/2 signals, as shown in Figure 3a. Also, the existence of iron was clearly verified using energy-dispersive X-ray (EDX) spectroscopy (Figure S8a,b). After the B-AuNPs were capped, the signal of gold appeared (Figure S8b). The specific surface area (BET plot) of the as-synthesized WMSNs was 282 m2/g and it fell to 218 and 177 m2/g after the incorporation of ESIONPs and functionalization of N-(3triethoxysilylpropyl)gluconamide (Figure S9a). Although some 5813

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 3. (a) X-ray photoelectron spectroscopy of WMSNs, mS-ESIONPs@WMSNs, ligand-mS-ESIONPs@WMSNs, B-Ar-S-ligand-mSESIONPs@WMSNs, BAuNPs-ligand-mS-ESIONPs@WMSNs. (b) Release profiles of Safranin O dye in water from BAuNPs-ligand-mSESIONPs@WMSNs at pH 4.5 and 7. Release profiles of Safranin O dye in water from BAuNPs-ligand-mS-ESIONPs@WMSNs in the presence of 100 mM mannitol. (c) Pulsatile release profile for BAuNPs-ligand-mS-ESIONPs@WMSNs as a function of pH variation.

small size and poor crystallinity of the ESIONPs, we could still observe the XRD pattern of ESIONPs and thus examine the existence of ESIONPs (Figure S11a). The appearance of strong signal of (111) Bragg peak of BAuNPs from BAuNPs-ligandmS-ESIONPs@WMSNs validated the content of Au (Figure S11b). FTIR was also performed to verify the grafting of folic acid onto the as-synthesized nanocomposites. As shown in Figure S12, the FTIR spectrum of FA-PCA-BAuNPs-ligandmS-ESIONPs@WMSNs exhibits the characteristic peaks of FA at 1643, 1604, and 1503 cm−1, corresponding to CO vibration and/or benzene (conjugated double bonds), N−H, and heteroring (conjugated double bonds), respectively. Reversible Controlled Release Behavior Studies. To verify the controllable release functionality, we dispersed the BAuNPs-ligand-mS-ESIONPs@WMSNs in phosphate buffer solution at pH 7, in which the boronate ester bonds conjugated B-AuNPs and ligand-mS-ESIONPs@WMSNs remained intact and blocked the pores to prevent the model guest safranin O dye from being released. As can be seen in Figure 3b, at pH 7, little free drug is observed, suggesting no leakage of the entrapped molecules and thus good retention of the entrapped molecules by B-AuNPs. At pH 4.5, simulating the pH environment of endosomes/lysosomes, the acid induced hydrolysis of boronic esters to open the nanogates and hence the releasing of safranin O dye, as indicated by the increase in the absorption band at 520 nm. The rate of dye release depends on the pH value; in other words, the proton concentration

affected the rate of hydrolysis of boronic esters and desorption of the drug, which had electrostatic interaction with the wall of the silica channel. For demonstration, Figure S13 shows the rapid release of dye to 100% within 12 min at pH 2−3, indicating the fast hydrolyzation of boronate ester bonds and fast dye desorption at low pH value. The reversible characteristic of the boronate ester bonds is essential for the on−off behavior of the gatekeepers. To prove this concept, we dispersed the BAuNPs-ligand-mS-ESIONPs@ WMSNs nanocomposites into a phosphate buffer solution of pH 7 and held the mixture for 10 min. Obviously, as shown in Figure 3c, the drug release was inhibited due to the capping of BAuNPs so the release percentage increased negligibly. Upon sudden increase acidity to pH 3, the entrapped safranin O was released rapidly, indicating that the capping particles were switched to an open position. Once the pH was adjusted back into 7 at 15 min, the boronate ester bond formed again, switching onto the capping position and stopping the release of the drug. The switch cycle was repeated several times, and similar release responses were observed. Note that the release gradients of each switching decreases, which indicates that less of the drug was released than the previous one. This can be rationalized by the amount remaining inside the mesopore which was reduced each time as the drug was released into the surrounding, resulting in the decrease of amount of the drug released and hence the diffusion rate (Figure 3c). 5814

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 4. (a) Prussian blue staining of BAuNPs-ligand-mS-ESIONPs@WMSN-treated HeLa cells under optical microscopy. (b) Fielddependent magnetization curves (at 300 K) of ligand-mS-ESIONPs@WMSNs and BAuNPs-ligand-mS-ESIONPs@WMSNs. (c) CT images of BAuNPs-ligand-mS-ESIONPs@WMSNs with serial dilution. From left to right: [Au] = 20, 15, 10, 5, 2.5 mg/mL. (d) In vitro T1-weighted images of serial diluted nanoparticle-ingested HeLa cells. From left to right: [Fe] = 1.0, 0.5, 0.25, 0 (control) mM. (e) In vitro T1-weighted images of mesenchymal stem cells after ingestion of nanoparticles at different concentration. From left to right: [Fe] = 1.0, 0.5, 0.25, 0 (control) mM.

The boronate ester bond can also be hydrolyzed by cis-diols. In this approach, we used mannitol, an FDA-approved cis-diol for diuresis. As clearly shown in Figure 3b, in the presence of 100 mM mannitol, the release behavior was similar to that at pH 4.5. Mannitol has higher affinity with boronic acid in the physiological condition and would compete with saccharidederivative polyalcohols ligands on the surface of mesoporous silica nanoparticles, leading to the hydrolysis of the boronate ester bond between boronic acid and N-(3triethoxysilylpropyl)gluconamide. Clinically, mannitol has been widely used for reducing high intracranial pressure via systemic intravenous injection after a satisfactory serum level is achieved. We thus propose that mannitol can also be delivered as an efficient triggering agent to release chemotherapy agents once the nanocomposites are accumulated in the targeted tumor sites. Cellular Uptake and Relaxivity Property. MTT assay was then used for viability tests. In the cases of ligand-mSESIONPs@WMSNs and BAuNPs-ligand-mS-ESIONPs@ WMSNs, compared with the untreated control, there was no significant reduction in the cell viability of HeLa cells in concentrations ranging from 20 to 150 ppm after 24 h of incubation with these nanoparticles. Instead, the viabilities of the cells had increased by 2−8% as the concentration increased, suggesting that the cytotoxicity of the NCs was negligible. The similar tendency of ligand-mS-ESIONPs@WMSNs and BAuNPs-ligand-mS-ESIONPs@WMSNs might be attributed to the contribution of the saccharide coating anchored on the surface of mS-ESIONPs@WMSNs. Saccharide and its derivatives consist of a sugar unit and have proven to be very biocompatible.44 We also verified that the saccharide coating could indeed improve the biocompatibility of our nanoparticles (Figure S14a,b). To confirm the cellular uptake of the as-synthesized nanoparticles containing ESIONPs, we performed a Prussian

blue stain experiment. After 15 h of incubation, we stained the cells with potassium hexacyanoferrate(II) solution and characterized the cells with optical microscopy. The intracellular blue color clearly implied the formation of iron(II, III) hexacyanoferrate(II, III), providing clear evidence that the nanoparticles containing Fe3+ were indeed ingested by the HeLa cells (Figure 4a and Figure S15a). We further verified the properties of MR imaging of the as-synthesized nanocomposites. Iron oxide nanoparticles are more biocompatible than gadolinium-based and manganese-based materials because iron species are abundant component of hemoglobin in red blood cell. The as-synthesized extremely small iron oxide nanoparticles of about 2 nm had negligible magnetization and thus had lower r2 values, which caused a low r2/r1 ratio. Considering their biocompatibility, increased r1 value, and lower r2/r1 ratio, ESIONPs appear to be suitable for use as a series of good T1 contrast agents. The combination of ESIONPs and mesoporous silica nanoparticles provides a drug delivery system with T1-weighted MR imaging. We have then verified the relaxation properties of the nanocomposites with different concentrations of iron oxide nanoparticles inside (for assessing the concentration, see Supporting Information). The results are listed in Figure S15b of the Supporting Information. According to the Solomon−Bloembergen−Morgan (SBM) theory, the relaxation enhancement originates from both innersphere (IS) and outer-sphere (OS) mechanisms. The overall longitudinal relaxivity is thus the sum of the inner-sphere (IS), r1IS, and outer-sphere (OS) mechanisms, r1OS. Factors that affect inner-sphere mechanism are the number of fast exchanging water molecules in the first coordination sphere of the metal ions, the residence time of the inner-sphere water molecules, and the tumbling time of the metal ion, while the outer-sphere mechanism is mainly affected by the sum of the diffusion coefficient of bulk water and the metal ions. As the 5815

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 5. Confocal microscopic images of HeLa cells incubated with DOX loaded BAuNPs-ligand-mS-ESIONPs@WMSNs for 2, 6, and 16 h. The cell cytoskeleton was stained with FITC (green). The nuclei were stained with DAPI (blue). The merged image represents FITC + DAPI + DOX emission.

content of ESIONPs increases, r1 values increase as well. This is due to the increase of metal ions and thus the increase of the number of fast exchanging water molecules in the first coordination sphere of the metal ions that contribute to r1IS. The combination of ESIONPs and mesoporous silica slows down the diffusion rate, and hence, the diffusion coefficient would be lower. The lower diffusion coefficient results in the enhancement of r1OS. The contribution of both r1IS and r1OS leads to a higher r1 value. However, r2 would also be affected by the diffusion coefficient; therefore, the r2 value would slightly increase as well. Note that a suitable T1 contrast agent should have high r1 and low r2/r1. It is thus important to finely control the content of ESIONPs so that r1 value is high while r2 value remains unchanged or increases only slightly in this approach. In addition, we also examined the field-dependent magnetization curves (at 300 K) of ligand-mS-ESIONPs@WMSNs and BAuNPs-ligand-mS-ESIONPs@WMSNs to show rather small magnetization of our materials (Figure 4b). In Vitro Cell Imaging Studies. The imaging effect of the as-synthesized nanocomposites with serial dilution of this drug delivery system was demonstrated. ESIONPs encapsulated inside WMSNs contributed a T1-weighted MRI contrast effect (Figure S15c), while the BAuNPs acting as gates that capped the surface of the WMSNs exhibited X-ray absorption that contributed to the computer tomography (CT) contrast effect (Figure 4c). We also performed the MR images in vitro by feeding B-AuNPs-ligand-mS-ESIONPs@WMSN to HeLa cells for 3 h to allow the particles to accumulate in endosomes/ lysosomes via endocytosis. MR imaging showed that the collected cell pellets exhibited T1 contrast capability at different

iron concentrations, which ensured that the T1 signals still existed after ingestion by cells (Figure 4d). The significant increase in signal intensity with the increase in iron content demonstrates its ability for cell trafficking. A similar tendency can also be achieved in the case of mesenchymal stem cells (Figure 4e), indicating the potential application of our nanocomposites for stem cell trafficking. Of the various type of imaging technologies applied to early diagnosis and clinical usage, CT provides high-resolution 3D structural details of tissues, but the low-contrast medium sensitivity limits its applicability. In contrast, MRI exhibits higher contrast sensitivity and is ionizing-free. The combination of these two imaging techniques is superior to using individually in terms of contrast sensitivity and resolution, making this system appealing for further clinical application. Intracellular Drug Delivery Studies. The controlled release platform of the nanocomposite was then demonstrated by releasing doxorubicin, a well-known chemotherapeutic agent toward malignancy, from BAuNPs-ligand-mS-ESIONPs@ WMSNs. The encapsulated amount of doxorubicin was determined to be ∼100 mg of doxorubucin per gram of the as-synthesized nanoparticles by comparing the difference in absorbance of DOX between initial amount and residue in supernatants, followed by the calculation based on the calibration curve according to absorbance versus DOX concentration. Laser scanning confocal microscopy was used to verify the internalization of the released DOX from BAuNPsligand-mS-ESIONPs@WMSNs nanocomposites. We treated HeLa cells with DOX within BAuNPs-ligand-mS-ESIONPs@ WMSNs, as shown in Figure 5. From the experimental results, 5816

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Figure 6. (a) Optical microscopy images of HeLa (upper panel) and A549 (lower panel) cells incubated with FA-PCA-BAuNPs-ligand-mSESIONPs@WMSNs. All the experiments followed the sequence with 4 °C/20 min incubation → 37 °C/24 h culture. After 20 min incubation at 4 °C, the untargeted nanoparticles were removed by PBS wash. Confocal microscopic images of HeLa (left panel) and A549 (right panel) cells incubated with FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSNs. All the experiments followed the sequence with 4 °C/20 min incubation → 37 °C/24 h culture. After 20 min incubation at 4 °C, the untargeted nanoparticles were removed by PBS wash. The nuclei were stained with DAPI. The merged image represents DAPI + DOX emission. (HeLa: (b) DAPI, (c) bright field, (d) DOX, (e) merge. A549: (f) DAPI, (g) bright field, (h) DOX, (i) merge.) Confocal microscopic images of HeLa cells incubated with DOX loaded FA-PCA-BAuNPsligand-mS-ESIONPs@WMSNs. The endosomes and lysosomes were stained with (i) LysoTracker Blue and (j) LysoTracker Green. The merged image represents LysoTracker + DOX emission. 5817

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

endosomes/lysosomes were stained with LysoTracker Blue and Green. The results shown in Figure 6j,k, indicate that DOX red signal was mainly localized with endosome/lysosome tracker while some was not. It has been commonly accepted that the ingestion of nanoparticles by cells is through endocytosis intracellular delivery pathway. Since the controlled release of our nanoparticles is triggered under acidic condition, the appearance of DOX red signal inside endosomes/lysosomes should come from DOX released from FA-PCA-BAuNPsligand-mS-ESIONPs@WMSN. We thus conclude the existence of drug delivery nanoparticles inside endosomes/lysosomes. Moreover, a small portion of DOX is expected to escape from the endosomes/lysosomes to the cytosol, which accounts for their nonoverlap with the endosome/lysosome tracker. For clarity, the Z-stack confocal images are also shown in Figure S16 to ensure the distribution of DOX inside endosomes/ lysosomes. In our current study, once the nanocarriers were exposed in the acidic endosomes/lysosomes, the DOX release was triggered and the confocal images showed that at 2 h the DOX fluorescence was first located at the nuclei (Figure 5), while in Figure 6, after incubation for 24 h, most DOX molecules accumulated at the cytoplasm. When nanoparticles are introduced into biological media, the adsorption of proteins or small molecules existing in biological fluids onto the surface of nanoparticles, forming multilayered biomolecules, called protein corona, can act as a shield to impede the drug release and improve carrier properties for drug delivery.48,49 We thus believe the different DOX delivery rate inside HeLa cells before and after functionalizing protocatechuic acid and folic acid onto the drug delivery nanoparticles results from the adsorption of protein corona, which interacts with surface functionalized ligands and varies from case to case, to certain extent, also plays a role of controlled release gate in addition to boronic acid functionalized gold nanoparticles. In other words, for pHcontrolled release, protein corona on nanoplatform may actually block the release of drug at neutral pH, while at endosomes/lysosomes, corona-protected nanoplatforms also release drug after protein degradation, and this is the reason for affecting DOX delivery rate.50 For a drug carrier, intended to reduce side effects by having specificity to designated locations, it is necessary to have both controlled release and targeting abilities. Our drug delivery platform presented above certainly fits this criterion and proves the concept of integrating biomedical imaging (MRI/CT), cancer targeting, and controlled release of therapeutic agents into a nanocomposite. In a preliminary test for animal studies (n = 2), the T1-weighted images of FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSNs were monitored using a 7T animal micro MRI system as the NPs were administrated by intravenous injection of 1 mg Fe/ kg. The images were then captured at preinjection and postinjection after 20 min. The results shown in Figure S17 reveal T1-weighted signal enhancement accumulated in some tissues such as desired tumor part and liver where we believe the metabolism of the nanoparticles exists. However, a number of obstacles must be overcome before the current integrated drug delivery platforms can have practical clinical applications. Although injection of nanoparticles intravenously is often used in preclinical animal studies, some reports about the in vivo biodistribution of nanoparticles showed that a large proportion of injected nanoparticles would be captured by liver and spleen during circulation.51−54 Although the enhanced permeability and retention effect makes nanoparticles easier to accumulate in tumors, the proportion is still rather low due to

the DOX emission signal appeared in the nuclei after 2 h of incubation. The emissions of DAPI and DOX were almost overlapping in position, indicating the release of doxorubicin into the nuclei. The internalization of nanoparticles occurred mainly through endocytosis,45 and the particles were trapped inside endosomes, late endosomes, and lysosomes, where the environment is acidic (pH = 4−5).46 Once the BAuNPs-ligandmS-ESIONPs@WMSNs nanoparticles were exposed to these acidic compartments inside the cancer cells, the boronate ester bond hydrolyzed rapidly and caused the capping particles to open and release the chemotherapeutic agent. The emergence of the DOX signal in nuclei after 2 h of incubation provides evidence that pH is indeed a suitable and effective trigger. The increased intensity of the fluorescence emission in cytoplasm and nuclei could be seen as the incubation time increased (Figure 5), indicating that the uptake and the accumulation of doxorubicin were time-dependent. We further confirmed the cell viability after treatment with DOX-loaded BAuNPs-ligandmS-ESIONPs@WMSNs with MTT assay. As expected, the cell viability decreased as the concentration of DOX fed to HeLa cells increased, indicating that the doxorubicin loaded in the BAuNPs-ligand-mS-ESIONPs@WMSNs released from the channels of the mesopore, entering the nucleus to react with DNA and hence induced apoptosis (Figure S14c). Intracellular Targeting Studies. Cellular imaging of folic acid targeting and intracellular drug delivery was further demonstrated. We chose human cervical epithelial carninoma cell line (HeLa, FR-positive) and human lung adenocarcinoma epithelial cell line (A549, FR-negative) to inspect the expression of folate receptors. To compare the targeting capability of FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSNs bound to the cell surfaces of HeLa and A549, we fed the nanoparticles to the cells and incubated them at 4 °C, a temperature known to retard vesicular trafficking pathways, for 20 min.47 The untargeted nanoparticles were removed by PBS wash and fresh medium was added, followed by additional culture at 37 °C for 24 h to allow the targeting nanoparticles bound on the cell surface to be ingested into the cells via receptor-mediated endocytosis. As shown in Figure 6, in the case of HeLa cells, after further incubation for 24 h, the DOX red signal was mainly localized in the cytoplasm, and a small amount of the signal was in the nuclei. The emission of DOX red signals in cytoplasm confirmed the internalization of FAPCA-BAuNPs-ligand-mS-ESIONPs@WMSNs and the targeting ability of our drug delivery system with the help of FA. In comparison, there was no obvious DOX signal in the malignant lung A549 cells after the same experimental procedure. This lack of a signal might be ascribed to the low FR expression of A549, which resulted in much less nanoparticle uptake (Figure 6). This result supports the idea that the current system has high selectivity toward cells with the targeted receptors. We also used optical microscopy to characterize the cell density difference before and after targeting particles in HeLa and A549 cell lines. There was little change in the case of A549, indicating little uptake of DOX loaded FA-PCA-BAuNPs-ligand-mSESIONPs@WMSNs which was removed by PBS wash after being held at 4 °C for 20 min (Figure 6a). In the case of HeLa cells, however, cell density obviously decreased and cell shapes were abnormal after 24 h of incubation relative to the control set, indicating that DOX-loaded particles indeed affected the growth of the cells. To further confirm that nanoparticles are trapped inside endosomes/lysosomes, we fed FA-PCABAuNPs-ligand-mS-ESIONPs@WMSN to HeLa and the 5818

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

METHODS

the phagocytic activity of the Kupffer cells in reticuloendothelial systems. For a suitable contrast agent applied in vivo, the challenges are to provide high concentration of contrast agent in the blood to obtain a stable concentration of this contrast agent medium in the blood for a sufficiently long time as well as to be able to use passive or active targeting of these contrasts for tissue or tumor characterization. There have been some studies about multimodality imaging probes designed during the past few years in order to allow high-resolution, highsensitivity investigation of biological activity.55,56 However, the combination of these imaging functions without sacrificing their individual properties is still a challenge owing to the short circulation time which nanoparticles would be rapidly captured by RES system and then cleared from the bloodstream via kidney. Since the destination of the nanocarriers is tumor, and the effective controlled release therapy and image enhancement depend on the concentration of nanoparticles, sufficient accumulation volume in tissue or tumor is necessary. Although some reports claimed that ligands such as polyethylene glycol (PEG) onto nanoparticles could increase their residence time in blood, their elimination by the RES system is still a major obstacle. Recently, some researches applied the direct intratumoral injection which might improve both tumor concentrations and the tumor-to-organ ratios of nanoparticles compared to intravenous injection. Intratumoral injection can thus open the possibility to promote the chemotherapy efficacy and imaging enhancement due to the improvement of nanoparticles accumulation volume in tumors. Further in vivo tests of our theranostic platform and the improvement of using intratumoral injection are an ongoing project. Particularly, the metabolic pathway of nanoparticles and the proportion of particle accumulation in different tissue will require more intensive investigations.

Synthesis of ESIONPs@WMSN Nanoparticles. In a typical procedure, 30 μL of the ESIONPs in 1 mL of chloroform was dispersed in 10 mL of CTAB solutions (0.1 M) and sonicated for more than 2 h until the chloroform evaporated, leaving transparent pale yellow ESIONPs/CTAB solution. Then, 30 mg of as-synthesized WMSNs was poured, which is dispersed in 1 mL of water into the ESIONPs/CTAB solution. After the mixture stirred for 20 min, 0.04 g of NH4F and 35 μL of tetraethylorthosilicate (TEOS) were added into the solution, and the mixture was kept at 80 °C for 1 h. The assynthesized mS-ESIONPs@WMSNs were washed 2 times with ethanol to remove the unreacted species, and the mixture was dispersed in 10 mL of ethanol. Surface Modification of mS-ESIONPs@WMSN Nanoparticles with N-(3-Triethoxysilylpropyl)gluconamide. An ethanol solution of mS-ESIONPs@WMSNs was mixed with 100 μL of N-(3triethoxysilylpropyl)gluconamide. The mixture was stirred for 15 h at room temperature to induce the hydrolyzation and condensation of silane ligand on the surface of mS-ESIONPs@WMSN. The resulting ligand-mS-ESIONPs@WMSN NPs were collected after washing them with water for more than 3 times to remove any unreacted N-(3triethoxysilylpropyl)gluconamide. Finally, the surfactant (CTAB) was removed via the ion exchange method, in which the products were transferred to 10 mL of water containing 0.5 g of NH4NO3 and the solution was maintained at 80 °C for 2 h. The extraction step was repeated twice to ensure the removal of the surfactants. Synthesis of B-AuNPs Capped Ligand-mS-ESIONPs@WMSN Nanoparticles. To obtain the hybrid solid that functions as a molecular gate, a portion of 30 mg of ligand-mS-ESIONPs@WMSN NPs was added to a suspension of 1 mg of B-AuNPs in 2 mL of DMSO, and the mixture was stirred for 15 h. The resulting solid BAuNPs capped ligand-mS-ESIONPs@WMSN nanoparticles were then washed with water several times and then dispersed with 1 mL of DMSO. Synthesis of FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSN. To graft folic acid onto BAuNPs-ligand-mS-ESIONPs@WMSN, we use 3,4-dihydroxybenzoic acid (protocatechuic acid, PCA) as the linker. The as-prepared 3,4-dihydroxybenzoic acid solution (0.0124 g in 1 mL of DMSO) was added into BAuNPs-ligand-mS-ESIONPs@ WMSN solution and the solution reacted for 5 h. The amino groups of FA were modified with carboxylate groups of PCA to form amide bonds through EDC/HNS chemistry. A DMSO solution of EDC (10 mM, 1 mL), NHS (10 mM, l mL), and FA (2 mM, 1 mL) was mixed with PCA-BAuNPs-ligand-mS-ESIONPs@WMSN for 15 h. The collected precipitates by centrifugation of colloidal solutions were washed with DMSO three times to remove excess FA. Preparation of Dox-Loaded BAuNPs-ligand-mS-ESIONPs@ WMSN Nanoparticles. The as-synthesized ligand-mS-ESIONPs@ WMSN nanoparticles were dispersed in 1 mL of DMSO and mixed with 1 mg of doxorubicin in 1 mL of DMSO at room temperature for 15 h, followed by addition of the as-synthesized BAuNPs and further stirring for 15 h. The resulting Dox-loaded BAuNPs-ligand-mSESIONPs@WMSN nanoparticles were then retrieved by repeating the procedures of centrifugation and dispersion in DMSO several times. The absorbance difference in Dox between the initial amount and residue in supernatants was performed to estimate the entrapped Dox concentration following a standard linear calibration curve based on the measurements of Dox absorbance. In Vitro Release of Dye via Change of pH Condition. The loading of Safranine O into the mesopore was performed by mixing the dye solution with the ligand-mS-ESIONPs@WMSN for 1 day, so that dye would diffuse into the pore voids through the gradient of dye concentration inside and outside the pore. To study the controlled release behavior affected by different pH condition, 20 mg of safranine O loaded-BAuNPs-ligand-mS-ESIONPs@WMSNs was dispersed in 1 mL of aqueous solution with pH 4.5 or pH 7 to evaluate the gate-like effect via the study of dye release from the pore voids of the polyalcohol-functionalized materials at 37 °C. After the sample was centrifuged, the absorbance of the supernatant was measured by monitoring at 520 nm, where there is the maximum absorbance of

CONCLUSIONS In summary, this is the report of work on fabricating a targeted reversible controlled release drug delivery system with extremely small iron oxide nanoparticles exhibiting T1-weighted MR contrast capability to achieve both imaging and therapeutic purposes. The ultimate goal of a drug delivery system is to execute various missions in vivo. However, many impediments need to be overcome before application to more complicated biological systems such as the human body. Over the past few years, delivery vehicles with different functions have been developed and are still under intensive investigation. The incorporation of ESIONPs and capping of BAuNPs followed by the grafting of targeting molecules reported above sets a prototype for integrating a drug delivery platform consisting of ESIONPs and BAuNPS to exhibit T1-weighted MR and CT contrast enhancement. The dual imaging will contribute to more accurate diagnosis. For the transport of cargo inside organisms, we then combined a controlled release carrier capped with gold nanoparticles and targeting folic acid to prevent undesired leakage and nonspecific uptake. The strategically designed mesoporous silica based carrier thus presents a platform that is capable of delivering antitumor agents and other functional cargos to fulfill a range of uses or purposes. En route to the pragmatic diagnostic and therapeutic application in vivo, our system should demonstrate the development of ultimate integrated delivery platform. 5819

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

mL) and streptomycin (0.05 mg/mL) were incubated for 15 h with sample. After incubation, the culture medium was removed and the adherent cells were washed twice with PBS. For Prussian blue staining, an as prepared mixed solution of equal parts of 2 N hydrochloric acid and 2% potassium ferrocyanide solution was added, 1 mL per well. After 30 min, the supernatant was discarded and cells were washed with distilled water several times. The cell samples were observed with an optical microscope (OLYMPUS IX81). In Vitro MR Imaging. MRI was performed using a clinical 3 T MR System (Signal Excite, GE Healthcare). After treatment with particles for 3 h, the cell samples were washed with PBS and centrifuged in test tubes bathing in a homemade water tank. The tank was then placed in an 8 channel head coil. Two-dimension T1-weighted fast spin echo pulse sequences were used (TR/TE = 550/13 ms). The slice thickness was 1.0 mm with a 0.5 mm gap, and the field of view (FOV) was 14 × 10 cm2. The matrix size was 288 × 192. Total scan time was 4 min and 5 s at the NEX of 2. The images were then analyzed at a workstation provided by GE Healthcare (Advantage workstation 4.2). CT. For evaluation of imaging capability of BAuNPs-ligand-mSESIONPs@WMSN in computed tomography, BAuNPs-ligand-mSESIONPs@WMSNs were placed in an Eppendorf tube with serial dilution. The tubes were positioned in a homemade rack that was filled with water. We placed the rack in a Sixty-four Multislice CT (Lightspeed VCT, GE Healthcare). The rack was scanned 4 times under 80 keV, 100 mA at the field of view (FOV) of 32 cm. The resolution was 512 × 512 and the slice thickness was 0.625 mm. Under this condition, a voxel is 0.625 × 0.625 × 0.625 cm3, which is isotropic. The CT numbers of each test tube were measured at the workstation provided by the vendor of CT (Advantage Workstation AW 4.2_07, GE Healthcare).

Safranin O dye to characterize the delivery of the Safranin O dye from the pore voids to the aqueous solution. In Vitro Release of Dye via cis-Diol. The loading of Safranine O into the mesopore was performed by mixing the dye solution with the ligand-mS-ESIONPs@WMSN for 1 day, so that dye would diffuse into the pore voids through the gradient of dye concentration inside and outside the pore. To study the controlled release behavior affected by the addition of cis-diol 20 mg of Safranine O loaded-BAuNPs-ligandmS-ESIONPs@WMSN was dispersed in 1 mL of aqueous solution with the concentration of 100 mM mannitol to evaluate the gate-like effect via the study of dye release from the pore voids of the polyalcohol-functionalized materials at 37 °C. After the sample was centrifuged, the absorbance of the supernatant was measured by monitoring at 520 nm to characterize the delivery of the Safranin O dye from the pore voids to the aqueous solution. Cell Images of HeLa Cells Treated with Dox-Loaded BAuNPs-ligand-mS-ESIONPs@WMSN Nanoparticles Using Confocal Laser Scanning Microscope (CLSM). To study the particles ingested via endocytosis, cells were cultured in DMEM at 37 °C supplied with 5% CO2/ 95% air. Cells were trypsinized and seeded onto 22 × 22 mm2 in a 6-well culture dish with 5 × 104 cells in each well. After 24 h of incubation, each well was washed twice with phosphate buffered saline (PBS), and then 0.2 mL of Dox-loaded BAuNPs-ligand-mS-ESIONPs@WMSN nanoparticles (100 μg/mL in DMEM) was added. After 2, 6, and 16 h of incubation, cells were washed with PBS and then fixed with 1% paraformaldehyde in PBS for 10 min. For the confocal fluorescence imaging, the cell cytoskeleton was stained with fluorescein isothiocyanate (FITC), and the nuclei were stained with 4′,-diamidino-2-phenylindole (DAPI). The cells treated with particles were subjected to a confocal laser scanning microscope (Zeiss LSM710 NLO) equipped with 63× (PAPO, 1.40 oil immersion) objective. A combination of 405 nm diode laser, 488 nm Argon laser, and 543 nm Argon laser was used as the excitation source for nucleus, cytoskeleton, and doxorubicin, respectively. In Vitro Cytotoxicity. The cell viability was analyzed by using a colorimetric assay agent, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Roche) as described (Mosmann 1983). The HeLa cells were seeded in a 96-well plate with 5 × 104 cells per well in a 90% Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum, penicillin (50 U/mL), and streptomycin (0.05 mg/mL). To contrast with the control, four different dosages of nanoparticles were added to each well: 20, 50, 100, and 150 μg/mL. After 15 h of incubation, wells were washed twice with PBS and then incubated with 200 μL of the culture medium with 10% MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) agent per well. After 4 h of reaction time, the culture medium was removed and replenished with 200 μL of dimethyl-sulfoxide (Merck) per well to dissolve the purple MTT formazan crystal. The optical density of these samples was measured at 595 nm. All measurements were done with three replicates using and ELISA reader (VersaMax Microplate Spectrophotometers; Molecular-Devices). In Vitro Targeting Study of HeLa and A549 Cells Treated with FA-PCA-BAuNPs-ligand-mS-ESIONPs@WMSN Using a Confocal Laser Scanning Microscope. Cells were cultured in DMEM at 37 °C supplied with 5% CO2/95% air. Cells were trypsinized and seeded in 6-well chamber slides with 5 × 104 cells in each well. Each well was washed twice with PBS after 24 h of incubation and then 2 mL of DOX loaded FA-PCA-BAuNPs-ligandmS-ESIONPs@WMSN was added. The cells were then treated with particles at 4 °C for 20 min. Then, the medium containing untargeted nanoparticles was removed and fresh medium was added, followed by the additional culture at 37 °C for 24 h to perform particle internalization into cells. After that, culture medium was removed and the sample was washed with PBS and then fixed using 1% paraformaldehyde for 20 min at room temperature. The nuclei were stained with DAPI and the cells treated with particles were subjected to a confocal laser scanning microscope for observation. Cell Labeling. Cells (5 × 104 cells per well in 6-well Falcon tissue culture plate) cultured in a 90% Dulbecco’s modified Eagle’s medium (DMEM; Sigma) containing 10% fetal bovine serum, penicillin (50 U/

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b08130. Part of experimental section, TEM, HRTEM, SEM, XPS, EDX, relaxivity properties, N2 adsorption−desorption analysis, XRD, FTIR, MTT assay, Prussian blue stained images, the estimation of the quantity of nanoparticles per cell, Z-stack confocal microscopic images, animal studies images, and confocal images (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2113-M-002-015-MY2 and MOST 102-2628-B-303-001-MY3). REFERENCES (1) Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Theranostic Nanoplatforms for Simultaneous Cancer Imaging and Therapy: Current Approaches and Future Perspectives. Nanoscale 2012, 4, 330−342. (2) Li, C. A Targeted Approach to Cancer Imaging and Therapy. Nat. Mater. 2014, 13, 110−115. (3) Argyo, C.; Weiss, V.; Brauchle, C.; Bein, T. Multifunctional Mesoporous Silica Nanoparticles As a Universal Platform for Drug Delivery. Chem. Mater. 2014, 26, 435−451. (4) Cheng, Z.; Zaki, A. A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities. Science 2012, 338, 903−910. 5820

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano

Dual Modal CT/MRI Molecular Imaging. J. Am. Chem. Soc. 2010, 132, 13270−13278. (24) Shanmugam, V.; Chien, Y. H.; Cheng, Y. S.; Liu, T. Y.; Huang, C. C.; Su, C. H.; Chen, Y. S.; Kumar, U.; Hsu, H. F.; Yeh, C. S. Oligonucleotides-Assembled Au Nanorod-Assisted Cancer Photothermal Ablation and Combination Chemotherapy with Targeted Dual-Drug Delivery of Doxorubicin and Cisplatin Prodrug. ACS Appl. Mater. Interfaces 2014, 6, 4382−4393. (25) Ruiz-Hernandez, E.; Baeza, A.; Vallet-Regı, M. Smart Drug Delivery through DNA/Magnetic Nanoparticle Gates. ACS Nano 2011, 5, 1259−1266. (26) Chang, Y. T.; Liao, P. Y.; Sheu, H. S.; Tseng, Y. J.; Cheng, F. Y.; Yeh, C. S. Near-Infrared Light-Responsive Intracellular Drug and siRNA Release Using Au Nanoensembles with OligonucleotideCapped Silica Shell. Adv. Mater. 2012, 24, 3309−3314. (27) Schlossbauer, A.; Warncke, S.; Gramlich, P. M. E.; Kecht, J.; Manetto, A.; Carell, T.; Bein, T. A Programmable DNA-Based Molecular Valve for Colloidal Mesoporous Silica. Angew. Chem., Int. Ed. 2010, 49, 4734−4737. (28) Vivero-Escoto, J. L.; Slowing, I. I.; Wu, C. W.; Lin, V. S. Y. Photoinduced Intracellular Controlled Release Drug Delivery in Human Cells by Gold-Capped Mesoporous Silica Nanosphere. J. Am. Chem. Soc. 2009, 131, 3462−3463. (29) Patel, K.; Angelos, S.; Dichtel, W. R.; Coskun, A.; Yang, Y. W.; Zink, J. I.; Stoddart, J. F. Enzyme-Responsive Snap-Top Covered Silica Nanocontainers. J. Am. Chem. Soc. 2008, 130, 2382−2383. (30) Park, C.; Oh, K.; Lee, S. C.; Kim, C. Controlled Release of Guest Molecules from Mesoporous Silica Particles Based on a pHResponsive Polypseudorotaxane Motif. Angew. Chem., Int. Ed. 2007, 46, 1455−1457. (31) Nguyen, T. D.; Leung, K. C. F.; Liong, M.; Pentecost, C. D.; Stoddart, J. F.; Zink, J. I. Construction of a pH-Driven Supramolecular Nanovalve. Org. Lett. 2006, 8, 3363−3366. (32) Hernandez, R.; Tseng, H. R.; Wong, J. W.; Stoddart, J. F.; Zink, J. I. An Operational Supramolecular Nanovalve. J. Am. Chem. Soc. 2004, 126, 3370−3371. (33) Xiao, D.; Jia, H. Z.; Zhang, J.; Liu, C. W.; Zhuo, R. X.; Zhang, X. Z. A Dual-Responsive Mesoporous Silica Nanoparticle for TumorTriggered Targeting Drug Delivery. Small 2014, 10, 591−598. (34) Zhang, J.; Yuan, Z. F.; Wang, Y.; Chen, W. H.; Luo, G. F.; Cheng, S. X.; Zhuo, R. X.; Zhang, X. Z. Multifunctional EnvelopeType Mesoporous Silica Nanoparticles for Tumor-Triggered Targeting Drug Delivery. J. Am. Chem. Soc. 2013, 135, 5068−5073. (35) Luo, Z.; Ding, X.; Hu, Y.; Wu, S.; Xiang, Y.; Zeng, Y.; Zhang, B.; Yan, H.; Zhang, B.; Yan, H.; et al. Engineering a Hollow Nanocontainer Platform with Multifunctional Molecular Machines for Tumor-Targeted Therapy in Vitro and in Vivo. ACS Nano 2013, 7, 10271−10284. (36) Guan, S.; Ge, D.; Liu, T. Q.; Ma, X. H.; Cui, Z. F. Protocatechuic Acid Promotes Cell Proliferation and Reduces Basal Apoptosis in Cultured Neural Stem Cells. Toxicol. In Vitro 2009, 23, 201−208. (37) Peng, Y. K.; Liu, C. L.; Chen, H. C.; Chou, S. W.; Tseng, W. H.; Tseng, Y. J.; Kang, C. C.; Hsiao, J. K.; Chou, P. T. Antiferromagnetic Iron Nanocolloids: A New Generation in Vivo T1 MRI Contrast Agent. J. Am. Chem. Soc. 2013, 135, 18621−18628. (38) Kim, J.; Kim, H. S.; Lee, N.; Kim, T.; Kim, H.; Yu, T.; Song, I. C.; Moon, W. K.; Hyeon, T. Multifunctional Uniform Nanoparticles Composed of a Magnetite Nanocrystal Core and a Mesoporous Silica Shell for Magnetic Resonance and Fluorescence Imaging and for Drug Delivery. Angew. Chem., Int. Ed. 2008, 47, 8438−8441. (39) Liong, M.; Lu, J.; Kovochich, M.; Xia, T.; Ruehm, S. G.; Nel, A. E.; Tamanoi, F.; Zink, J. I. Multifunctional Inorganic Nanoparticles for Imaging, Targeting, and Drug Delivery. ACS Nano 2008, 2, 889−896. (40) Kim, J.; Lee, J. E.; Lee, J.; Yu, J. H.; Kim, B. C.; An, K.; Hwang, Y.; Shin, C. H.; Park, J. G.; Kim, J.; et al. Magnetic Fluorescent Delivery Vehicle Using Uniform Mesoporous Silica Spheres Embedded with Monodisperse Magnetic and Semiconductor Nanocrystals. J. Am. Chem. Soc. 2006, 128, 688−689.

(5) Park, K. Facing the Truth about Nanotechnology in Drug Delivery. ACS Nano 2013, 7, 7442−7447. (6) Lu, J.; Liong, M.; Zink, J. I.; Tamanoi, F. Mesoporous Silica Nanoparticles As a Delivery System for Hydrophobic Anticancer Drugs. Small 2007, 3, 1341−1346. (7) Chen, W.; Tsai, P. H.; Hung, Y.; Chiou, S. H.; Mou, C. Y. Nonviral Cell Labeling and Differentiation Agent for Induced Pluripotent Stem Cells Based on Mesoporous Silica Nanoparticles. ACS Nano 2013, 7, 8423−8440. (8) Duan, R.; Xia, F.; Jiang, L. Constructing Tunable Nanopores and Their Application in Drug Delivery. ACS Nano 2013, 7, 8344−8349. (9) Tao, Z.; Toms, B.; Goodisman, J.; Asefa, T. Mesoporous Silica Microparticles Enhance the Cytotoxicity of Anticancer Platinum Drugs. ACS Nano 2010, 4, 789−794. (10) Caravan, P. Strategies for Increasing the Sensitivity of Gadolinium Based MRI Contrast Agents. Chem. Soc. Rev. 2006, 35, 512−523. (11) Caravan, P.; Ellison, J. J.; McMurry, T. J.; Lauffer, R. B. Gadolinium(III) Chelates As MRI Contrast Agents: Structure, Dynamics, and Applications. Chem. Rev. 1999, 99, 2293−2352. (12) Ananta, J. S.; Godin, B.; Sethi, R.; Moriggi, L.; Liu, X.; Serda, R. E.; Krishnamurthy, R.; Muthupillai, R.; Bolskar, R. D.; Helm, L.; et al. Geometrical Confinement of Gadolinium-Based Contrast Agents in Nanoporous Particles Enhances T1 Contrast. Nat. Nanotechnol. 2010, 5, 815−821. (13) Taylor, K. M. L.; Kim, J. S.; Rieter, W. J.; An, H.; Lin, W.; Lin, W. Mesoporous Silica Nanospheres As Highly Efficient MRI Contrast Agents. J. Am. Chem. Soc. 2008, 130, 2154−2155. (14) Alric, C.; Taleb, J.; Duc, G. L.; Mandon, C.; Billotey, C.; MeurHerland, A. L.; Brochard, T.; Vocanson, F.; Janier, M.; Perriat, P.; et al. Gadolinium Chelate Coated Gold Nanoparticles As Contrast Agents for Both X-ray Computed Tomography and Magnetic Resonance Imaging. J. Am. Chem. Soc. 2008, 130, 5908−5915. (15) Bridot, J. L.; Faure, A. C.; Laurent, S.; Rivière, C.; Billotey, C.; Hiba, B.; Janier, M.; Josserand, V.; Coll, J. L.; Elst, L. V.; et al. Hybrid Gadolinium Oxide Nanoparticles: Multimodal Contrast Agents for in Vivo Imaging. J. Am. Chem. Soc. 2007, 129, 5076−5084. (16) Werner, E. J.; Datta, A.; Jocher, C. J.; Raymond, K. N. HighRelaxivity MRI Contrast Agents: Where Coordination Chemistry Meets Medical Imaging. Angew. Chem., Int. Ed. 2008, 47, 8568−8580. (17) Shin, J.; Anisur, R. M.; Ko, M. K.; Im, G. H.; Lee, J. H.; Lee, I. S. Hollow Manganese Oxide Nanoparticles As Multifunctional Agents for Magnetic Resonance Imaging and Drug Delivery. Angew. Chem., Int. Ed. 2009, 48, 321−324. (18) An, K.; Kwon, S. G.; Park, M.; Na, H. B.; Baik, S. I.; Yu, J. H.; Kim, D.; Son, J. S.; Kim, Y. W.; Song, I. C.; et al. Synthesis of Uniform Hollow Oxide Nanoparticles through Nanoscale Acid Etching. Nano Lett. 2008, 8, 4252−4258. (19) Cheong, S.; Ferguson, P.; Feindel, K. W.; Hermans, I. F.; Callaghan, P. T.; Meyer, C.; Slocombe, A.; Su, C. H.; Cheng, F. Y.; Yeh, C. S.; et al. Simple Synthesis and Functionalization of Iron Nanoparticles for Magnetic Resonance Imaging. Angew. Chem., Int. Ed. 2011, 50, 4206−4209. (20) Wang, Y.; Ng, Y. W.; Chen, Y.; Shuter, B.; Yi, J.; Ding, J.; Wang, S. C.; Feng, S. S. Formulation of Superparamagnetic Iron Oxides by Nanoparticles of Biodegradable Polymers for Magnetic Resonance Imaging. Adv. Funct. Mater. 2008, 18, 308−318. (21) Jang, J. T.; Nah, H.; Lee, J. H.; Moon, S. H.; Kim, M. G.; Cheon, J. Critical Enhancements of MRI Contrast and Hyperthermic Effects by Dopant-Controlled Magnetic Nanoparticles. Angew. Chem., Int. Ed. 2009, 48, 1234−1238. (22) Kim, B. H.; Lee, N.; Kim, H.; An, K.; Park, Y. I.; Choi, Y.; Shin, K.; Lee, Y.; Kwon, S. G.; Na, H. B.; et al. Large-Scale Synthesis of Uniform and Extremely Small-Sized Iron Oxide Nanoparticles for High-Resolution T1 Magnetic Resonance Imaging Contrast Agents. J. Am. Chem. Soc. 2011, 133, 12624−12631. (23) Chou, S. W.; Shau, Y. H.; Wu, P. C.; Yang, Y. S.; Shieh, D. B.; Chen, C. C. In Vitro and in Vivo Studies of FePt Nanoparticles for 5821

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822

Article

ACS Nano (41) Li, Y.; Xiao, W.; Xiao, K.; Berti, L.; Luo, J.; Tseng, H. P.; Fung, G. Well-Defined, Reversible Boronate Crosslinked Nanocarriers for Targeted Drug Delivery in Response to Acidic pH Values and cisDiols. Angew. Chem., Int. Ed. 2012, 51, 2864−2869. (42) Geng, J.; Li, M.; Wu, L.; Chen, C.; Qu, X. Mesoporous Silica Nanoparticle-Based H2O2 Responsive Controlled-Release System Used for Alzheimer’s Disease Treatment. Adv. Healthcare Mater. 2012, 1, 332−336. (43) Shi, P.; Li, M.; Ren, J.; Qu, X. Gold Nanocage-Based Dual Responsive “Caged Metal Chelator” Release System: Noninvasive Remote Control with Near Infrared for Potential Treatment of Alzheimer’s Disease. Adv. Funct. Mater. 2013, 23, 5412−5419. (44) Gan, Q.; Lu, X.; Yuan, Y.; Qian, J.; Zhou, H.; Lu, X.; Shi, J.; Liu, C. A Magnetic, Reversible pH-Responsive Nanogated Ensemble Based on Fe3O4 Nanoparticles-Capped Mesoporous Silica. Biomaterials 2011, 32, 1932−1942. (45) Chen, Z.; Li, Z.; Lin, Y.; Yin, M.; Ren, J.; Qu, X. Biomineralization Inspired Surface Engineering of Nanocarriers for pH-Responsive, Targeted Drug Delivery. Biomaterials 2013, 34, 1364− 1371. (46) Zhang, X.; Yang, P.; Dai, Y.; Ma, P.; Li, X.; Cheng, Z.; Hou, Z.; Kang, X.; Li, C.; Lin, J. Multifunctional Up-Converting Nanocomposites with Smart Polymer Brushes Gated Mesopores for Cell Imaging and Thermo/pH Dual-Responsive Drug Controlled Release. Adv. Funct. Mater. 2013, 23, 4067−4078. (47) Chien, Y. H.; Chou, Y. L.; Wang, S. W.; Hung, S. T.; Liau, M. C.; Chao, Y. J.; Su, C. H.; Yeh, C. S. Near-Infrared Light Photocontrolled Targeting, Bioimaging, and Chemotherapy with Caged Up-Conversion Nanoparticles in Vitro and in Vivo. ACS Nano 2013, 7, 8516−8528. (48) Pino, P.; Pelaz, B.; Zhang, Q.; Maffre, P.; Nienhaus, G. U.; Parak, W. J. Protein Corona Formation Around Nanoparticles − From the Past to the Future. Mater. Horiz. 2014, 1, 301−313. (49) Cifuentes-Rius, A.; de Puig, H.; Kah, J. C. Y.; Borros, S.; HamadSchifferli, K. Optimizing the Properties of the Protein Corona Surrounding Nanoparticles for Tuning Payload Release. ACS Nano 2013, 7, 10066−10074. (50) Behzadi, S.; Serpooshan, V.; Sakhtianchi, R.; Muller, B.; Landfester, K.; Crespy, D.; Mahmoudi, M. Protein Corona Change the Drug Release Profile of Nanocarriers: The “Overlooked” Factor at the Nanobio Interface. Colloids Surf., B 2014, 123, 143−149. (51) He, Q.; Zhang, Z.; Gao, F.; Li, Y.; Shi, J. In Vivo Biodistribution and Urinary Excretion of Mesoporous Silica Nanoparticles: Effects of Particle Size and PEGylation. Small 2011, 7, 271−280. (52) Pan, L.; Liu, J.; He, Q.; Shi, J. MSN-Mediated Sequential Vascular-to-Cell Nuclear-Targeted Drug Delivery for Efficient Tumor Regression. Adv. Mater. 2014, 26, 6742−6748. (53) Ling, D.; Park, W.; Park, S.; Lu, Y.; Kim, K. S.; Hackett, M. J.; Kim, B. H.; Yim, H.; Jeon, Y. S.; Na, K.; et al. Multifunctional Tumor pH-Sensitive Self-Assembled Nanoparticles for Bimodal Imaging and Treatment of Resistant Heterogeneous Tumors. J. Am. Chem. Soc. 2014, 136, 5647−5655. (54) Wu, Z. C.; Li, W. P.; Luo, C. H.; Su, C. H.; Yeh, C. S. RattleType Fe3O4@CuS Developed to Conduct Magnetically Guided Photoinduced Hyperthermia at First and Second NIR Biological Windows. Adv. Funct. Mater. 2015, 25, 6527−6537. (55) Louie, A. Multimodality Imaging Probes: Design and Challenges. Chem. Rev. 2010, 110, 3146−3195. (56) Dong, W.; Li, Y.; Niu, D.; Ma, Z.; Liu, X.; Gu, J.; Zhao, W.; Zheng, Y.; Shi, J. A Simple Route to Prepare Monodisperse Au NPDecorated, Dye-doped, Superparamagnetic Nanocomposites for Optical, MR, and CT Trimodal Imaging. Small 2013, 9, 2500−2508.

5822

DOI: 10.1021/acsnano.5b08130 ACS Nano 2016, 10, 5809−5822