Biocompatibility of Calcined Mesoporous Silica Particles with

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Biocompatibility of Calcined Mesoporous Silica Particles with Cellular Bioenergetics in Murine Tissues Mariam Al Shamsi,† Mohammed T. Al Samri,‡ Suhail Al-Salam,§ Walter Conca,| Sami Shaban,⊥ Sheela Benedict,‡ Saeed Tariq,∆ Ankush V. Biradar,#,∇ Harvey S. Penefsky,O Tewodros Asefa,*,#,∇ and Abdul-Kader Souid*,‡ Departments of Immunology, Pediatrics, Pathology, Internal Medicine, Medical Education, and Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates UniVersity, Al Ain, Abu Dhabi, 17666, United Arab Emirates, Department of Chemistry and Chemical Biology, 610 Taylor Road, and Department of Chemical Engineering and Biochemical Engineering, 98 Brett Road, Rutgers, The State UniVersity of New Jersey, Piscataway, New Jersey 08854, United States, and Department of Biochemistry, Public Health Research Institute, 225 Warren Street, Newark, New Jersey 07103, United States ReceiVed July 24, 2010

A novel in vitro system was developed to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues. O2 consumption by lung, liver, kidney, spleen, and pancreatic tissues was unaffected by exposure to 200 µg/mL MCM41cal or SBA15-cal for several hours. Normal tissue histology was confirmed by light microscopy. Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy. The results show reasonable in vitro biocompatibility of the mesoporous silicas with murine tissue bioenergetics. Introduction The potential adverse effects of nanoparticles and micromaterials on human health are of great concern and contemporary interest (1-3). Silica (SiO2) is one of the most abundant materials on earth and is widely used in many areas. Silica microparticles and nanoparticles can be cytotoxic or harmless, depending mainly on their polymorph type as well as their other structural parameters such as surface area, surface composition, etc. (4-28). Exposure to crystalline forms of silica particles, typically quartz, is well-known to cause serious diseases, such as lung cancer, autoimmune disorders, renal impairment, silicosis, autoimmune disease, and oxidative stress via surfacederived free radical generation and intracellular production of reactive oxygen species (ROS) (4-17). Thus, quartz and related crystalline forms of silica are classified by the International Agency for Research on Cancer (IARC) in Class 1. However, with few exceptions of materials such as vitreous silicasa form of amorphous silicasand others (18-20), many forms of amorphous silicas are not very pathogenic and have been classified by IARC in Class 3 (21-25). Recent toxicology studies of mesoporous silicas, which are also amorphous forms of silica with nanoscale sizes and nanoporous structure, have revealed contradictory findings regarding the materials’ toxicity (29-34). While high doses of * To whom correspondence should be addressed. (A.-K.S.) Tel: 009713-973429. Fax: +971-3-973422. E-mail: [email protected]. (T.A.) Tel: 732-445-2970. Fax: 732-445-5312. E-mail: [email protected]. † Department of Immunology, United Arab Emirates University. ‡ Department of Pediatrics, United Arab Emirates University. § Department of Pathology, United Arab Emirates University. | Department of Internal Medicine, United Arab Emirates University. ⊥ Department of Medical Education, United Arab Emirates University. ∆ Department of Anatomy, United Arab Emirates University. # Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey. ∇ Department of Chemical Engineering and Biochemical Engineering, Rutgers, The State University of New Jersey. O Public Health Research Institute.

mesoporous silicas administered subcutaneously to mice are reported to be relatively harmless, the same doses given intravenously or intraperitonealy are shown to be lethal (29). Other cellular studies involving mesoporous silicas on cancer cells showed similar contradictory results (29-35). These different reports on the biological activities for mesoporous materials can be the result of their different surface areas, surface silanol groups, surface composition, cellular uptakes, and particle sizes and shapes (26-28, 42). For instance, the degree of hydroxyl radical formation by silica particles in solution and its subsequent biological effect and cytotoxicity depend on the particles’ structure and surface properties (42). On the other hand, silica nanoparticles, including mesoporous silicas, are emerging as candidate materials for drug or gene delivery (31-41). Thus, in light of their potential importance and conflicting reports on their biocompatibility, further investigation of toxicity of mesoporous silica in animal models is warranted. In general, our knowledge of the fate of mesoporous silicas in various tissues does not match with the rapid pace of research on their syntheses in numerous forms and structures. In particular, the impact of composition, method of synthesis, residual material, surface charges, size, and shape on their biological activities and toxicities remains relatively unexplored while different synthetic conditions are known to produce mesoporous materials with significant structural and morphological variations. The term cellular respiration implies delivery of O2 and metabolic fuels to the mitochondria, oxidation of reduced fuels with passage of electrons to O2, and synthesis of ATP. Impaired respiration thus entails an interference with any of these processes. We recently described the toxicity of two commonly studied mesoporous silica particles (solvent-extracted MCM41 and SBA-15) in HL-60 (myeloid) and Jurkat (lymphoid) cells (43). As compared to MCM-41 (regular spherical- and ovalshaped), SBA-15 particles (irregular rod-shaped) exhibit greater lattice spacing, bigger Barrett-Joyner-Halenda (BJH) pore diameters, and smaller Brunauer-Emmett-Teller (BET) surface

10.1021/tx100245j  2010 American Chemical Society Published on Web 10/20/2010

Biocompatibility of Calcined Mesoporous Silica Particles

areas. SBA-15 inhibited respiration in both cell lines. MCM41, on the other hand, was not toxic. However, both particles delayed the onset of glucose-driven respiration in cells depleted of metabolic fuels and inhibited respiration in isolated mitochondria (43). We present here in vitro measurements on cellular respiration (mitochondrial O2 consumption) of murine tissues exposed to calcined MCM-41 or SBA-15 (MCM41-cal or SBA15-cal). The primary purpose of the study was to investigate whether calcined mesoporous silicas are biocompatible with cellular bioenergetics, that is, whether mesoporous silica nanoparticles affect the biochemical cellular processes related to energy transformations in our body including cellular respirations, metabolic processes, and production and utilization of ATP and energy. In addition, the impact of postsynthetic procedures (calcination vs solvent extraction of the templates) on tissue respiration was examined (see the Supporting Information). Solvent-extracted mesoporous silica particles, which often contain residual synthetic materials, inhibited pneumatocyte respiration. In contrast, calcination produced particles that were free of residual synthetic materials such as surfactants and were biocompatible with tissues. On the basis of previous studies and some of our results, some differences in composition and surface chemistry between the two materials, that is, prepared by calcination or solvent extraction, are to be expected. These include differences such as density of surface silanols and residual surfactants in the materials, which we believe may have contributed to the differences in toxicity and biological activity of the two types of materials (26-28, 42).

Experimental Procedures Materials and Methods. Pd(II) complex of meso-tetra-(4sulfonatophenyl)-tetrabenzoporphyrin, sodium salt (Pd phosphor) was obtained from Porphyrin Products (Logan, UT). Complete Protease Inhibitor Cocktail was purchased from Roche Applied Science (Indianapolis, IN). Poly(ethylene oxide)-block-poly-(butylene oxide)-block-poly(ethylene oxide) (P123) was purchased from BASF. Bovine serum albumin (free of endotoxins and fatty acids), cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), and remaining reagents were supplied by SigmaAldrich (St. Louis, MO). Solutions. Pd phosphor (2.5 mg/mL ) 2.0 mM) was prepared in distilled water (dH2O) and stored at -20 °C. NaCN (1.0 M) was prepared in dH2O, and the pH was adjusted to ∼7.0 with 12 N HCl. Glucose oxidase (10 mg/mL) was prepared in dH2O and stored at -20 °C. Complete Protease Inhibitor Cocktail was prepared by dissolving one tablet in 1.0 mL of dH2O and stored at -20 °C. Synthesis of Calcined and Solvent-Extracted MCM-41 (MCM41-cal and MCM41-ext). First, as-synthesized MCM-41 was prepared as described previously (43). Briefly, 11 mmol of CTAB was dissolved in 960 mL of Millipore water and 14 mL of 2.0 M NaOH solution. After the solution was stirred moderately at 80 °C for 30 min, 101.2 mmol of TEOS was added. The resulting solution was stirred for 2 h at 80 °C. The hot solution was then filtered, and the solid product was washed with Millipore water (4 × 80 mL), followed by ethanol (4 × 80 mL). The material was then dried in oven at 80 °C. These procedures resulted in as-synthesized mesostructured MCM-41. The surfactant template from the as-synthesized MCM-41 was removed by calcination. The calcination procedure was done by keeping 2 g of the as-synthesized MCM-41 sample in a tube furnace at 550 °C for 6 h in the flow of air (the temperature was raised from room temperature to 550 °C at a ramp of 10 °C/min). The calcined MCM-41 particles, labeled as MCM41-cal, were then obtained.

Chem. Res. Toxicol., Vol. 23, No. 11, 2010 1797 For solvent extraction, 2 g of the as-synthesized MCM-41 was suspended in a mixture of 1 mL (12.1 N) of HCl and 600 mL of anhydrous ethanol and stirred at 50 °C for 6 h. The resulting solution was filtered, and the solid sample was washed with copious amounts of Millipore water and ethanol (3 × 80 mL in each case) and dried at ambient conditions. These steps gave solvent-extracted MCM41 particles (MCM41-ext). Synthesis of Calcined and Solvent-Extracted SBA-15 (SBA15-cal and SBA15-ext). First, as-synthesized SBA-15 was prepared as reported with minor modifications (44). A solution containing 12 g of Pluronics [(PEO)20(PPO)70(PEO)20, BASF], 313 g of Millipore water, and 72 g of HCl (36 wt %) was prepared and stirred vigorously at 40-45 °C until the Pluronics template dissolved. TEOS (25.6 g) was added, and the stirring continued at 45 °C for 24 h. The solution was then kept in oven at 80 °C without stirring for 24 h. The hot solution was filtered, and the solid product on filter paper was washed with Millipore water and ethanol (3 × 80 mL in each case) and dried at ambient conditions. This procedure produced as-synthesized SBA-15 sample. The Pluronics surfactant was removed by calcination as above, giving calcined SBA-15 sample that is labeled as SBA15-cal. To remove the Pluronics surfactant by solvent extraction, 3 g of the sample was refluxed for 6 h in a solution containing 300 mL of ethanol and 300 mL of diethyl ether. The solution was filtered, and the solid phase was washed with water and ethanol (3 × 80 mL in each case) and dried at ambient conditions. This procedure gave solvent-extracted SBA-15 particles (SBA15-ext). The particles were stored at 25 °C in a Dry-Seal vacuum desiccator. The characteristics of MCM41-cal and SBA15-cal are compiled in Table 1, and their transmission electron micrographs (TEMs) are shown in Figure 1. The results indicate that these calcined mesoporous samples have highly ordered mesoporous structures, high surface areas, and almost no residual surfactant molecules in them. Tissues and Tissue Incubations. Lung, liver, spleen, kidney, and pancreatic tissues (7-30 mg pieces) were excised from the organs of an anesthetized (urethane, 25% solution, w/v, in 0.9% NaCl, using 100 µL per 10 g body weight) male BALB/c mouse using a 3-5 mm biopsy punch (Stiefel Laboratories, Inc., Research Triangle Park, NC) or scissors (Moria Vannas Wolg Spring, catalog no. ST15024-10). Specimens were immediately immersed in icecold Krebs-Henseleit buffer (115 mM NaCl, 25 mM NaHCO3, 1.23 mM NaH2PO4, 1.2 mM Na2SO4, 5.9 mM KCl, 1.25 mM CaCl2, 1.18 mM MgCl2, and 6 mM glucose, pH 7.4) saturated with O2: CO2 (95:5) prior to transferring them to 250 mL beakers containing 100 mL of Krebs-Henseleit buffer with and without silicate particles. For the pancreatic tissue, 2.0 µL per mL complete protease inhibitors was added. All procedures were in compliance with the guidelines of animal care (United Arab Emirates University, Faculty of Medicine and Health Sciences). Silica particles were dispersed in 100 mL of Krebs-Henseleit buffer and sonicated for about 10 s (using Branson 2210 Ultrasonics, Branson, MO) to disperse aggregates. Tissue specimens were then added, and the incubation continued at 37 °C with continuous gassing with 95% O2:5% CO2 (without agitation). For the pancreatic tissue, 2.0 µL per mL complete protease inhibitors was added to the incubation solution. At indicated time periods, specimens were removed from the incubation solution, rinsed thoroughly with Krebs-Henseleit buffer, weighed, and placed in 1 mL of Pd phosphor solution (Krebs-Henseleit buffer containing 0.5% albumin and 3 µM Pd phosphor) for O2 measurement. Solid-State 29Si MAS NMR Spectra. 29Si MAS solid-state NMR spectra of the mesoporous materials were acquired using a 300 MHz Bru¨ker Avance NMR spectrometer. The measurements were done with a 7.0 kHz spin rate, 100 s recycle delay, π/6 pulse width of 1.9 µs, and 1000 scans using high-power continuous-wave 1H decoupling. Light Microscopy. Pieces of lung, liver, spleen, kidney, and pancreas were fixed in 10% neutral formalin, dehydrated in increasing concentrations of ethanol, cleared with xylene, and

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Table 1. Physical Properties of the Studied Calcined Mesoporous Silica Particles (MCM41-cal and SBA15-cal) analytical methods powder X-ray diffraction nitrogen gas adsorption 13

C CP-MAS solid-state NMR spectroscopy elemental analysis

MCM41-cal

SBA15-cal

highly ordered mesostructures, sharp low-angle Bragg reflections type IV isotherms with steep capillary condensations, confirming mesoporous structures with large surface areas and uniform size mesopore sizes calcined samples: no residual surfactants or block copolymer; no residual ethoxy groups from TEOS no residual surfactants

29

very significant Q4 silicon sites at 110 ppm; very little Q3 and Q2 centered at 101 and 91 ppm, respectively

very significant Q4 silicon sites centered at 110 ppm; significant Q3 centered at 101 ppm and some Q1 centered at 91 ppm

TEM

regular spherical and oval-shaped particles with sizes ranging from 600 to 1000 nm in diameter

irregular-shaped particles of varying sizes (mostly rod-shaped with ∼400 to 500 nm diameter and ∼1000 nm long)

powder X-ray diffraction nitrogen gas adsorption nitrogen gas adsorption

lattice spacing ) ∼44 Å BET surface area ) ∼1290 m2/g (MCM41-cal) average BJH pore diametera ) ∼28 Å

lattice spacing ) ∼106 Å BET surface area ) ∼955 m2/g (SBA15-cal) average BJH pore diametera ) ∼114 Å

Si MAS solid-state NMR spectroscopy

a

Obtained from the desorption branch of gas adsorption isotherm.

Figure 1. TEM images of MCM41-cal (A and B) and SBA15-cal (C and D).

embedded in paraffin. Three micrometer sections were prepared from paraffin blocks and stained with hematoxylin and eosin. Staining for apoptosis was performed using the avidin-biotin immunoperoxidase method to detect activated caspase 3 (Cell Signaling Technology, Boston, MA). The staining was performed on 5 µm paraffin sections, using rabbit anticleaved caspase 3 antibodies. Positive and negative control sections for apoptosis were used. Electron Microscopy. Lung, liver, kidney, spleen, and pancreatic specimens were immersed in McDowell and Trump fixative for 3 h at room temperature (45). After they were rinsed with phosphate buffer, the tissues were postfixed with 1% osmium tetroxide for 1 h. After they were washed with dH2O, the samples were dehydrated in a series of graded ethanol and propylene oxide, infiltrated, and embedded in Agar-100 epoxy resin and polymerized at 65 °C for 24 h. Blocks were trimmed, and semithin and ultrathin sections were cut with Reichert Ultracuts, ultramicrotome. The semithin sections (130 µm) were stained with 1% aqueous toluidine blue on glass slides. The ultrathin sections (95 nm) on 200 mesh Cu grids were contrasted with uranyl acetate followed by lead citrate double stain (46, 4). The grids were studied/examined and photographed under a Philips CM10 transmission electron microscope. Oxygen Measurement. A phosphorescence oxygen analyzer was used to monitor O2 consumption (48). O2 was measured with the

Pd phosphor, which had an absorption maximum at 625 nm and a phosphorescence maximum at 800 nm. The samples were exposed to light flashes (600 times per min) from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc., Wheeling, IL). Emitted phosphorescent light was detected by a Hamamatsu photomultiplier tube (#928) after first passing it through a wide-band interference filter centered at 800 nm. The amplified phosphorescence decay was digitized at 1.0 MHz by a 20 MHz A/D converter (Computer Boards, Inc.). A program was developed using Microsoft Visual Basic 6, Microsoft Access Database 2007, and Universal Library components (Universal Library for Measurements Computing Devices, http:// www.mccdaq.com/daq-software/universal-library.aspx). It allowed a direct reading from the PCI-DAS 4020/12 I/O Board (PCI-DAS 4020/12 I/O Board, http://www.mccdaq.com/pci-data-acquisition/ PCI-DAS4020-12.aspx). Pulse detection was accomplished by searching for 10 phosphorescence intensities greater than 1.0 V (by default). Peak detection was accomplished by searching for the highest 10 data points of a pulse and choosing the data point closest to the pulse decay curve (49). The phosphorescence decay was exponential. The values of 1/τ were linear with dissolved [O2]: 1/τ ) 1/το + kq[O2], where 1/τ ) the phosphorescence decay rate in the presence of O2, 1/τo ) the phosphorescence decay rate in the absence of O2, and kq ) the secondorder O2 quenching rate constant in s-1 µM-1 (46). The instrument was calibrated with β-glucose and glucose oxidase (5). Respiration was measured at 37 °C. For each run, 1.0 mL of Pd phosphor solution (Krebs-Henseleit buffer containing 0.5% albumin and 3 µM Pd phosphor) was placed in a 1 mL glass vial. A piece of the tissue was added, and the vial was sealed with a crimp top aluminum seal. Mixing was with the aid of a parylene-coated stirring bar. The rate of respiration (zero-order, k, in µM O2 min-1) was set as the negative of the slope of a plot of [O2] vs time. The addition of 10 mM NaCN caused k ) d[O2]/dt to decrease almost to zero, confirming the decline in [O2] with time was mainly due to mitochondrial O2 consumption. The addition of glucose oxidase depleted remaining O2 in the solution. In Figure S1 in the Supporting Information, 29Si MAS NMR spectra of the MCM41-cal and SBA15-cal materials are displayed. The spectra show that both materials have prominent peaks corresponding to Q4 peaks. This result has been compiled with other structural information in Table 1. In Figure S2A in the Supporting Information, lung specimens (8-24 mg each) were incubated at 37 °C for up to ∼7.5 h with and without 25 µg/mL SBA15-cal. Rates of respiration (in µM O2 min-1 mg-1) are shown in Figure S2B in the Supporting Information. The rate (mean ( SD) of untreated samples (n ) 3) was 0.27


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( 0.04 µM O2 min-1 mg-1 and of treated samples (n ) 4) was 0.29 ( 0.04 µM O2 min-1 mg-1. In Figure S3 in the Supporting Information, liver specimens (7-10 mg each) were incubated at 37 °C for up to 5 h with and without 200 µg/mL MCM41-cal or SBA15-cal. Rates of respiration of untreated samples were 0.23 and 0.30, of sample treated with MCM41-cal 0.24 µM O2 min-1 mg-1, and of sample treated with SBA15-cal 0.30 µM O2 min-1 mg-1. Figure S4A in the Supporting Information shows rat lung respiration with and without 5 µg/mL MCM-41 with residual synthetic materials. The rate of respiration in untreated sample was linear with time (k ) 3.5 µM O2/min, R2 ) 0.932). The respiration of treated sample was nonlinear (R2 > 0.790) and was inhibited. The same experiment was repeated with 25 µg/ mL MCM-41 with residual synthetic materials (Figure S4B in the Supporting Information). The rate of respiration in untreated sample was linear with time (k ) 1.7 µM O2/min, R2 ) 0.947). The rate of respiration in treated sample was nonlinear (R2 > 0.772) and was inhibited. The same findings were also observed for SBA-15 with residual synthetic materials. Moreover, the same inhibition was noted in mouse lung and human lymphocytes, using MCM-41 or SBA-15 with residual synthetic materials. Thus, particles with residual synthetic materials are potent inhibitors of respiration.

Results and Discussion Synthesis of Mesoporous Materials and Procedure for Cell Studies. As-synthesized MCM-41 and SBA-15 were prepared as previously described (12). The surfactant templates were removed by calcinations (see the Experimental Procedures). The properties of the particles are shown in Figure 1, Figure S1 in the Supporting Information, and Table 1. To study the effect of silica particles on tissues, we developed an in vitro system to monitor respiration, using a phosphorescence O2 analyzer (45). Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer saturated with 95% O2:5% CO2. Specimens were incubated at 37 °C in the same buffer with continuous gassing with O2:CO2 (95:5) and without agitation. At specific time points, specimens were placed in Krebs-Henseleit buffer containing 0.5% albumin and 3 µM Pd phosphor for O2 measurement at 37 °C. [O2] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/τ were linear with [O2]: 1/τ ) 1/τ° + kq [O2]; 1/τ° ) the decay rate for zero O2, kq ) the rate constant in s-1 µM-1 (46). The addition of NaCN inhibited O2 consumption, confirming that oxidation occurred in the mitochondrial respiratory chain. Tissue samples incubated with 200 µg/mL MCM41-cal or SBA15cal for 5 h were also processed for light and electron microscopy (see details of the procedures in the Experimental Procedures). MCM41-cal Is Biocompatible with Alveolar Cell Respiration. Figure 2A,B shows alveolar cell (pneumatocyte) respiration with and without 200 µg/mL MCM41-cal. In Figure 2A, rates of respiration, k, for untreated and treated samples were similar over the 11 h incubation period. The values of k for untreated samples at hours 4 and 10 were 0.25 and 0.22 µM O2 min-1 mg-1, respectively. The corresponding rate for treated samples (mean ( SD, n ) 3) was 0.21 ( 0.0 µM O2 min-1 mg-1. The same experiment was repeated (Figure 2B). Over 11 h, the value of k for untreated samples (n ) 4) was 0.27 ( 0.03 µM O2 min-1 mg-1 and for treated samples (n ) 4) was 0.26 ( 0.06 µM O2 min-1 mg-1 (p ) 0.842). Similar findings were obtained in a third

Figure 2. Two independent experiments (A and B) of pneumatocyte (alveolar cells of lung specimens) respiration with and without 200 µg/mL MCM41-cal. The rate of respiration (k) was set as negative of the slope of [O2] vs time; the values of k (in µM O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of the particles. U, untreated; and T, treated.

independent experiment (data not shown). These data demonstrate biocompatibility of the calcined MCM41 particles with pneumatocyte respiration. In contrast, solvent-extracted MCM-41 particles (with residual synthetic materials) inhibited lung respiration (Figure S4A,B in the Supporting Information). MCM41-cal and SBA15-cal Are Biocompatible with Hepatocyte Respiration. Figure 3A,B shows alveolar cell respiration with and without 200 µg/mL SBA15-cal. In Figure 3A, rates of respiration for untreated and treated samples were similar over the 14 h incubation period. The value of k for untreated samples (n ) 5) was 0.22 ( 0.02 µM O2 min-1

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Figure 4. Liver tissue respiration with and without 200 µg/mL MCM41cal or SBA15-cal. Rates of respiration (in µM O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of the particles.

Figure 3. Two independent experiments (A and B) of pneumatocyte respiration with and without 200 µg/mL SBA15-cal are shown. The rate of respiration (k) was set as negative of the slope of [O2] vs time; the values of k (in µM O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of the particles. U, untreated; and T, treated.

mg-1 and for treated samples (n ) 5) was 0.15 ( 0.05 µM O2 min-1 mg-1 (p ) 0.035). The same experiment was repeated (Figure 3B). Over 12 h, the value of k for untreated samples (n ) 4) was 0.27 ( 0.03 µM O2 min-1 mg-1 and for treated samples (n ) 5) was 0.27 ( 0.06 µM O2 min-1 mg-1 (p ) 0.907). In Figure S2 in the Supporting Information, rates of respiration for untreated samples and for samples treated with 25 µg/mL SBA15-cal were similar over 7.5 h

(p ) 0.574). These data demonstrate reasonable biocompatibility of the SBA15-cal particles with pneumatocyte respiration. Biocompatibility of Calcined MCM41-cal and SBA15-cal Synthesized with Spleen, Kidney, and Pancreas Tissues. Figure 4 shows hepatocyte respiration with and without 200 µg/mL MCM41-cal or SBA15-cal. The rate of respiration for untreated sample was 0.32 µM O2 min-1 mg-1 (28 < t < 40 min), for sample treated with MCM41-cal 0.30 µM O2 min-1 mg-1 (60 < t < 82 min) and for sample treated with SBA15cal 0.28 µM O2 min-1 mg-1 (100 < t < 130 min). The same experiment was repeated (Figure S3 in the Supporting Information). Rates of respiration for untreated samples at minutes 81 and 287 were 0.23 and 0.30 µM O2/min, respectively. The corresponding rate for MCM41-cal treated sample was 0.24 µM O2 min-1 mg-1, and for SBA15-caltreated sample was 0.30 µM O2 min-1 mg-1. These results demonstrate biocompatibility of MCM41-cal and SBA15cal with hepatocyte respiration. Figure 5A,D shows respiration of spleen, kidney, and pancreatic tissues with and without 200 µg/mL MCM41-cal or SBA15-cal. In Figure 5A, the rate of respiration for untreated spleen was 0.29 µM O2 min-1 mg-1 (14 < t < 43 min) and for spleen treated with MCM41-cal was 0.28 µM O2 min-1 mg-1 (130 < t < 180 min). The rate of respiration for untreated kidney was 0.25 µM O2 min-1 mg-1 (44 < t < 80 min) and for kidney treated with MCM41-cal was 0.28 µM O2 min-1 mg-1 (190 < t < 230 min). The rate of respiration for untreated pancreas was 0.30 µM O2 min-1 mg-1 (83 < t < 127 min) and for pancreas treated with MCM41-cal was 0.20 µM O2 min-1 mg-1 (235 < t < 280 min). This low rate of pancreas respiration at minute 235 could be due to tissue degradation by pancreatic enzymes. Hence, in subsequent experiments with pancreatic tissues, complete protease inhibitors (2.0 µL per mL) were added. Figure 5B shows kidney respiration with and without 200 µg/mL MCM41-cal or SBA15-cal. The rate of respiration for untreated kidney (n ) 3) was 0.38 ( 0.12 µM O2 min-1 mg-1.



Figure 5. Respiration of spleen, kidney, and pancreatic tissues in the presence or absence of MCM41-cal and SBA15-cal. (A) Spleen, kidney, and pancreatic tissue respiration with and without 200 µg/mL MCM41-cal. (B) Kidney tissue respiration with and without 200 µg/mL MCM41-cal (M) or SBA15-cal (S). (C) Spleen tissue respiration with and without 200 µg/mL SBA15-cal. (D) Pancreatic tissue respiration with and without 200 µg/mL SBA15-cal. Rates of respiration (in µM O2 min-1 mg-1) are shown at the bottom of the runs. Zero minute corresponds to the addition of particles. U, untreated; M, MCM41-cal; and S, SBA15-cal.

The corresponding rates for kidney treated with MCM41-cal were 0.54 and 0.36 µM O2 min-1 mg-1 and for kidney treated with SBA15-cal were 0.41 and 0.31 µM O2 min-1 mg-1. The experiment was repeated with similar results (not shown). Figure 5C shows spleen respiration with and without 200 µg/ mL SBA15-cal. The rates of respiration for untreated spleen were 0.44 and 0.32 µM O2 min-1 mg-1 and for treated spleen were 0.27 and 0.29 µM O2 min-1 mg-1. Figure 5D shows pancreatic tissue respiration with and without 200 µg/mL SBA15-cal. The rate of respiration for untreated pancreas (n ) 3) was 0.40 ( 0.07 µM O2 min-1 mg-1

and for treated pancreas (n ) 2) was 0.27 ( 0.03 µM O2 min-1 mg-1 (p ) 0.07). In other experiments, rates of respiration for untreated pancreas were 0.40 and 0.35 µM O2 min-1 mg-1 and for pancreas treated with 200 µg/mL MCM41-cal were 0.33 and 0.30 µM O2 min-1 mg-1 (p ) 0.07). Tissue Histology and Electron Microscopy Imaging. Normal liver histology was observed in untreated samples as well as in samples treated with 200 µg/mL MCM41-cal or SBA15cal for 5 h (Figure 6). Furthermore, the staining for cleaved caspase 3 as a marker for apoptosis was negative (Figure 7). Similar preservation of normal architecture and histology as

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Figure 6. Light microscopy images. Liver specimens were incubated with and without silica particles for 5 h. The samples were then rinsed with Krebs-Henseleit buffer and stained with hematoxylin and eosin. Left, 40×; right, 100×. (A and B) Liver histology of untreated sample; (C and D) sample treated with 200 µg/mL MCM41-cal; and (E and F) sample treated with 200 µg/mL SBA15-cal.

well as negative staining for caspase 3 were obtained with treated lung, spleen, kidney, and pancreatic tissues (not shown). TEMs demonstrated abundant intracellular localization of particles in the studied tissues. Figure 8 shows representative images of lung (A) and liver (B) samples treated for 5 h with 200 µg/mL MCM41-cal and SBA15-cal, respectively. The particles were confined to the cytoplasm near the nucleus. Comparative Discussion. We studied two forms of mesoporous silica (MCM41-cal and SBA15-cal), which have potential applications as drug delivery vehicles. Our aim was to investigate their biocompatibility with cellular bioenergetics, especially since there have been conflicting reports on their toxicity (29-35, 50). A recent study reported the safety of mesoporous silica in vivo (50). In a conflicting report, Kohane et al. described in vitro toxicity of MCM-41 and SBA-15 in mesothelial cells (29). Furthermore, in mice, the route of administration had a major impact on tissue toxicity. Subcutaneous injections were nontoxic (29). Intraperitoneal and intravenous injections were highly toxic; lung examination revealed intravascular clots (29). In another study, 15 and 46 nm silica (>10 µg/mL for 42-72 h) reduced the viability of cultured bronchoalveolar carcinoma-derived

cells (6). Other cellular studies involving mesoporous silicas on cancer cells also showed contradictory results (29-35). Other noted toxicities were production of ROS, lipid peroxidation, and membrane damage (6). Silica nanoparticles can induce mitochondrial oxidative stress, including reactive O2 species, which deplete cellular glutathione (GSH) (6, 51-53). For instance, water-soluble fullerene nanoparticles are reported to transfer electrons to O2, producing reactive O2 species and lipid membrane peroxidation (54). Surface adsorptive properties of the particles were also thought to be a possible source of toxicity, especially since crystalline silica (Min-U-Sil 5) and coated (e.g., pegylated) silicas exhibited lower levels of toxicity (6, 55). It is also reported that the surface functional groups and composition, specific surface area, level of contaminants, and surface density of hydrophilic silanols of mesoporous materials can affect the cellular uptake and degree of hydroxyl radical formation in solution and the biological activity and cytotoxicity of silica particles (20, 26-28, 30, 42). Shapes of particles have long been known to affect the degree of particle agglomeration, circulation properties, and toxicity of several nonporous particles, including asbestos (56). Needle-shaped asbestos particles are known to be



Figure 7. Caspase 3 staining. Liver specimens were incubated with and without silica particles for 5 h. The samples were then rinsed with Krebs-Henseleit buffer and stained with anticleaved caspase 3 antibodies. (A) Positive control showing brown cytoplasmic staining (arrow) in apoptotic cells, (B) untreated sample, (C) sample treated with 200 µg/mL SBA15-cal, and (D) sample treated with 200 µg/mL MCM41-cal.

extremely toxic upon inhalation (56). Lin’s group has attempted to assess the effect of shape for mesoporous MCM41 materials by preparing different-shaped MCM-41 materials using different synthetic protocols (34, 57). Their results showed that cell membrane penetration for cancer cells and particle aggregations was correlated with the mesoporous particles’ morphologies. In our study, we observed that both materials showed biocompatible properties with various murine tissues once they are calcined, irrespective of their shapes. This indicates that the differences in shape of these two materials did not play a role to make the rod-shaped SBA15-cal any less biocompatible than the round- or ovalshaped MCM41-cal. It may also be because of the fact that the aspect ratio (length-to-width) of both materials is not large enough to result in any observable effects. Our investigation of the impact of postsynthetic procedures (calcination vs solvent extraction of the templates) on tissue respiration showed that solvent-extracted mesoporous silica particles containing residual synthetic materials inhibited pneumatocyte respiration (Supporting Information). In contrast, calcination produced particles that were free of residual synthetic materials and were biocompatible with tissues. On the basis of previous studies and some of our results, there are some expected differences in composition and surface chemistry between the two materials that are prepared by calcination and solvent extraction. We believe that some of these differences such as density of surface silanols and residual surfactant may have played roles in the differences in toxicity and biological activity between the two types of mesoporous silica materials, as also reported previously for other types of silica materials (26-28, 42). These differences include (1) different amounts of residual surfactants: the calcined materials have no residual surfactant while the solvent extracted ones almost always have at least trace amount of surfactant; (2) different density of surface hydroxyl (or silanols) groups: the calcined materials have less number

of surface silanols compared with the corresponding solventextracted samples because higher calcination temperature leads to condensation of surface silanols into siloxanes; (3) the differences in surface silanol density can further result in a difference in the materials’ ability to adsorb water on their surface: the solvent-extracted materials with more silanols are expected to adsorb more water because their better ability to form hydrogen-bonding interaction than the corresponding calcined materials that have less density of surface silanols; and (4) different average pore diameters: the calcined materials have slightly lower average pore diameter than the corresponding solvent-extracted samples, as a result of silanol condensation at higher temperature in the former. We believe that the differences in the density of surface silanols and the presence or absence of residual surfactants may have played major roles in the differences in toxicity and biological activity exhibited by the two types of materials. A similar observation has also been reported previously for other types of silica materials (26-28, 42, 51-53).

Conclusions In summary, we have shown here reasonable in vitro biocompatibility of MCM41-cal and SBA15-cal with cellular bioenergetics of various tissues (Table 2). This bioproperty could be attributed to a refined method of particle preparation suggesting residual surfactant molecules and also possibly the materials structure contributed to cytotoxicity (see the Supporting Information). MCM-41 and SBA-15 particles with surfactants extracted by solvent washing are not biocompatible with cells or tissues (Figure S4A,B in the Supporting Information) (12). The findings emphasize the impact of synthetic procedures on the cytotoxicity (or biocompatibility) of nanoparticles and the need for careful assessment of preparation protocols. Although both materials, that is, prepared by calcination and solvent extraction, are widely

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Al Shamsi et al.

lyzer used in the study here may be employed for analysis of cytotoxicity (or biocompatibility) of other forms of silica or other nanomaterials with great efficiency. Future studies will include obtaining additional information on the biocompatibility of calcined mesoporous silica particles on reticuloendothelial components of the featured organs, such as alveolar macrophages, dendritic cells, Kupffer cells, Mesangial cells, endothelial cells, etc. This may reveal further information regarding whether or not calcined mesoporous silica is able to generate ROS that could result in the end stage fibrosis. Acknowledgment. T.A. acknowledges the partial financial support by the U.S. National Science Foundation, CHE-0645348 and NSF DMR-0804846, for this research work. T.A. also thanks NSF for nominating him as one of its 10 NSF American Competitiveness and Innovation (NSF-ACI) Fellows for 2010 and the accompanying grant for his group’s research work. The work is also partially funded by United Arab Emirates University. The technical assistance by Manjusha Sudhadevi and Sarabjit Singh is greatly appreciated. Supporting Information Available: Figures of 29Si MAS NMR spectra, SBA15-cal biocompatible with alveolar cell respiration, MCM41-cal and SBA15-cal biocompatible with hepatocyte respiration, and MCM41-ext with residual synthetic materials. This material is available free of charge via the Internet at http://pubs.acs.org.

References

Figure 8. TEMs: Specimens were incubated with 200 µg/mL MCM41cal (A) or SBA15-cal (B) for 5 h. Samples were then rinsed with Krebs-Henseleit buffer and prepared for TEM. (A) TEM image showing intracytoplasmic round, electron-dense bodies (MCM41-cal particles, arrows) within a type 2 pneumatocyte, 28000×. (B) TEM image showing intracytoplasmic round, electron-dense bodies (SBA15cal particles, arrows) within a hepatocyte, 28000×.

Table 2. O2 Consumption by Murine Tissues with and without Silica Particles rate of respiration (µM O2 min-1 mg-1)a tissue

no addition

MCM41-cal

SBA15-cal

lung liver spleen kidney pancreas

0.24 ( 0.03 (28) 0.27 ( 0.13 (11) 0.28 ( 0.07 (10) 0.34 ( 0.12 (7) 0.35 ( 0.09 (10)

0.24 ( 0.05 (7) 0.21 ( 0.10 (3) 0.25 ( 0.06 (3) 0.31 ( 0.10 (3) 0.23 ( 0.09 (3)

0.21 ( 0.08 (10) 0.23 ( 0.10 (3) 0.26 ( 0.04 (3) 0.39 ( 0.13 (3) 0.24 ( 0.05 (4)

a Values are means ( SDs (n). The differences between the groups were not statistically significant.

considered for use in various applications, they have differences in composition and surface chemistry that are proven to be relevant for their biological differences as shown here. These include differences in the amount of residual surfactant, residual surface hydroxyl (or silanols) groups, average pore diameters, and tendency to adsorb water. We believe that the absence of residual surfactant as well as lower number of surface silanols in the calcined materials may have played roles in its biocompatibility. The phosphorescence O2 ana-

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