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Biocompatibility of Calcined Mesoporous Silica Particles with Ventricular Myocyte Structure and Function. Elhadi H. Aburawi†, Mohammed Anwar Qureshi...
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Biocompatibility of Calcined Mesoporous Silica Particles with Ventricular Myocyte Structure and Function Elhadi H. Aburawi,† Mohammed Anwar Qureshi,‡ Deniz Oz,‡ Petrilla Jayaprakash,‡ Saeed Tariq,§ Rashed S. Hameed,§ Sayantani Das,∥ Anandarup Goswami,∥,⊥ Ankush V. Biradar,∥,⊥ Tewodros Asefa,∥,⊥ Abdul-Kader Souid,† Ernest Adeghate,*,§ and Frank Christopher Howarth*,‡ Departments of †Pediatrics, ‡Physiology, 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 ABSTRACT: In vivo and in vitro systems were employed to investigate the biocompatibility of two forms of calcined mesoporous silica microparticles, MCM41-cal and SBA15-cal, with ventricular myocytes. These particles have potential clinical use in delivering bioactive compounds to the heart. Ventricular myocytes were isolated from 6 to 8 week male Wistar rats. The distribution of the particles in ventricular myocytes was investigated by transmission electron microscopy and scanning electron microscopy. The distribution of particles was also examined in cardiac muscle 10 min after intravenous injection of 2.0 mg/mL MCM41-cal. Myocyte shortening and the Ca2+ transient were determined following exposure to 200 μg/mL MCM41-cal or SBA15-cal for 10 min. Within 10 min of incubation at 25 °C, both MCM41-cal and SBA15-cal were found attached to the plasma membrane, and some particles were observed inside ventricular myocytes. MCM41-cal was more abundant inside the myocytes than SBA15-cal. The particles had a notable affinity to mitochondrial membranes, where they eventually settled. Within 10 min of intravenous injection (2.0 mg/mL), MCM41-cal traversed the perivascular space, and some particles entered ventricular myocytes and localized around the mitochondrial membranes. The amplitude of shortening was slightly reduced in myocytes superperfused with MCM41-cal or SBA15-cal. The amplitude of the Ca2+ transient was significantly reduced in myocytes superperfused with MCM41-cal but was only slightly reduced with SBA15-cal. Overall, the results show reasonable bioavailability and biocompatibility of MCM41-cal and SBA15-cal with ventricular myocytes.

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particles.6,7 For example, treatment of lymphocytes with multiwalled carbon nanotubes (20−40 nm) induces apoptosis.8 Silica and water-soluble fullerene nanoparticles extract electrons from the mitochondrial respiratory chain, producing deleterious oxidative damage.9−12 Many of these toxic properties are associated with ultrastructural cellular alterations.6 Moreover, changes in particle material, size, shape, surface area, aggregation, and conjugation may alter their biological effects. Therefore, a comprehensive study of the structure and function of these particles in cytoplasmic organelles is necessary before they can be used in clinical settings.

INTRODUCTION Mesoporous silicate of microparticles has potential clinical use in delivering bioactive compounds.1 The biological advantages of mesoporous microparticles stem from their large surface areas, microscale sizes, nanometer pores, and suitable properties for loading and delivering drugs and other bioactive molecules.1 They also, could be used in gene transfection and tissue engineering.2,3 Their channels can be used as a reservoir and can be opened or closed by different systems, e.g., polymers.2,3 Furthermore, the calcined mesoporous silica particles are bioavailable and biocompatible with various tissues, including the lung, liver, kidney, pancreas, and spleen.4,5 The term “nano-/micro-toxicology” implies structural or functional cellular derangements produced by 1 to 100 nm © 2012 American Chemical Society

Received: June 6, 2012 Published: December 13, 2012 26

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Table 1. Physical Characteristics of MCM41-cal and SBA15-cal analysis/ characterization transmission electron microscopy powder X-ray diffraction nitrogen gas adsorption 13 C CP-MAS solidstate NMR1 elemental analysis 29 Si MAS solid-state NMR nitrogen gas adsorption

MCM41-cal

SBA15-cal

regular spherical/oval shaped, 300−1,000 nm diameter

irregular shapes and sizes, mostly rods of 500 nm diameter and ∼1,000 nm long

highly ordered mesostructures with sharp low-angle Bragg reflection; lattice spacing of ∼44 Å type IV isotherms with steep capillary condensation; large surface area and mesopores of uniform size no residual cetyltrimethylammonium bromide surfactant (template); no residual ethoxy groups from TEOS no residual cetryltrimethylammonium bromide templates some Q4 silicon sites centered at ∼110 ppm; significant Q3 centered at ∼101 and some Q2 centered at ∼92 ppm BET surface area = ∼1290 m2/g; average BJH pore diameter = ∼28 Å

highly ordered mesostructures with sharp low-angle Bragg reflection; lattice spacing of ∼106 Å type IV isotherms with steep capillary condensations; large surface areas and mesopores of uniform size no residual pluronic P123 block copolymer templates; no residual ethoxy groups from TEOS no residual pluronic P123 block copolymer templates some Q4 silicon sites centered at ∼110 ppm; significant Q3 centered at ∼100 ppm and some Q2 centered at ∼93 ppm BET surface area = ∼955 m2/g; average BJH pore diameter = ∼59 Å

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The toxicities of two commonly studied solvent-extracted mesoporous silica particles Mobil Composition of Matter (MCM41-ext) and Santa Barbara Amorphous (SBA15-ext) have been investigated in HL-60 (myeloid) and Jurkat (lymphoid) cells.13 SBA15-ext (irregular rods averaging ∼500 nm in diameter and ∼1000 nm in length) inhibited respiration in both cell lines. MCM41-ext (spheres of ∼300 to 1000 nm in diameter), however, was nontoxic. Both particles, however, delayed the onset of glucose-driven respiration in cells depleted of metabolic fuels and inhibited respiration in isolated mitochondria.13 Doadrio et al. studied the application of SBA-15 as a gentamicin drug delivery system. The controlled release profile of gentamicin revealed evidence of initial burst release effect of 60%, and then showed a very slow release pattern.14 More recently, in vitro effects of calcined MCM-4l (labeled here as MCM41-cal) and SBA-15 (labeled here as SBA15-cal) were studied in murine tissues .4,5 The particles did not disturb cellular configuration or microorganelles. Because of their rigidity and surface charges, however, particles were firmly attached to (indenting) the plasma, nuclear, and mitochondrial membranes. Cellular respiration (cellular mitochondrial O2 consumption, a measure of cellular bioenergetics) was unaffected by incubating murine tissues (liver, lung, pancreas, kidney, and spleen) with 0.2 mg/mL MCM41-cal or SBA15-cal for 5 to 14 h. The results suggested reasonable bioavailability and biocompatibility of calcined mesoporous silicas in murine tissues. It has been shown that angiotensin II receptor (type 1) overexpression in infarcted hearts can be targeted by nanoparticlates.15 Other reports have demonstrated selective targeting of engineered nanoparticles to tumors.16,17 The feasibility of such targeting systems has been verified clinically.18 MCM41-cal and SBA15-cal, however, have not been thoroughly investigated in ventricular myocytes. These materials have imminent potential clinical use, and their effects on the heart muscle are an unmet need. The study here investigated MCM41-cal and SBA15-cal in rat ventricular myocytes. The purpose of this work was to evaluate their potential to deliver drugs, genes, or other bioactive compounds to the heart. The data overall support the findings in murine tissues, i.e., the bioavailability and biocompatibility of calcined mesoporous silicas in ventricular myocytes.

MATERIALS AND METHODS

Reagents. Type 1 collagenase was purchased from Worthington Biochemical Corp (Lakewood, NJ, USA). Fura 2-AM (F-1221) was purchased from Molecular Probes (Eugene, OR, USA). Pluronic P123 [poly(ethylene oxide)−poly(propylene oxide)−poly(ethylene oxide)(PEO20-PPO70-PEO20)] was purchased from BASF (Florham Park, NJ, USA). Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), fluorescein 5(6)-isothiocyanate, type XIV protease, and remaining reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Synthesis of Calcined MCM-41 Nanoparticles. Calcined MCM-41, MCM41-cal, was synthesized as described previously.4,19,20 Typically, CTAB (4.0 g, 1.1 mmol) was dissolved in Millipore water (960 mL) and 2.0 M NaOH solution (14 mL). After moderately stirring the solution at 80 °C for 30 min, TEOS (22.6 mL, 101.2 mmol) was added. The resulting solution was stirred for another 2 h at 80 °C. The hot solution was then filtered with a Whatman-1 filter paper, and the solid product was washed with Millipore water (4 × 80 mL), followed by ethanol (4 × 80 mL), and then dried in an oven at 80 °C. This resulted in mesostructured “as-synthesized MCM-41”. The CTAB surfactant template from the “as-synthesized MCM-41” was removed by calcination. Two grams of the “as-synthesized MCM41” sample was kept in a tube furnace at 550 °C for 6 h in airflow (temperature raised to 550 °C at a ramp of 10 °C/min). This produced calcined MCM-41 (labeled as MCM41-cal). Synthesis of Calcined SBA-15 Nanoparticles. Calcined SBA15, SBA15-cal, was prepared by some changes in molar concentration as described.4,19−21 A solution containing 12 g of Pluronic P123, 313 g Millipore water, and 72 g HCl (36 wt %) was prepared and stirred vigorously at 40 °C until the P123 template was dissolved. Then, TEOS (25.6 g, 0.123 mol) was added, and stirring of the solution was continued at 45 °C for 24 h. The solution was kept static in an oven at 80 °C for 24 h. The solid particles were then separated by filtration on Whatman-1 filter paper. The particles were washed with Millipore water and ethanol (3 × 80 mL, in each case) and dried at ambient conditions to produce “as-synthesized SBA-15” particles. The surfactant template was removed from “as-synthesized SBA-15” by calcination of 2 g of the “as-synthesized SBA-15” sample in a tube furnace at 600 °C for 6 h in air flow (temperature raised to 600 °C at a ramp of 10 °C/min), and the final product was named as SBA15-cal. The particles were stored at 25 °C in a vacuum desiccator until use. Characterization of MCM14-cal and SBA15-cal. The mesoporous MCM41-cal and SBA15-cal synthesized using surfactant supramolecular self-assembly method4,19−21 were characterized by various methods including nitrogen gas adsorption, elemental analysis, transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. The characterization results for MCM41-cal and SBA15-cal are compiled in Table 1. The surface area and pore diameters of the MCM41-cal and SBA15-cal were determined by N2 adsorption measurements which showed a type IV isotherm, indicating the presence of mesopores in them. The surface area and average BJH 27

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Figure 1. Large area and magnified TEM images of (A−C) MCM41-cal and (D−F) SBA15-cal and (G) representative 29Si MAS solid-state NMR spectrum of MCM41-cal material. pore diameter of MCM41-cal were found to be 1290 m2/g and 2.8 nm, respectively, whereas the surface area and average BJH pore diameter of SBA15-cal were 955 m2/g and 6.0 nm, respectively. TEM images of SBA15-cal and MCM41-cal are shown in Figure 1. The TEM images show that the MCM41-cal material possessed spherical or oval-shaped microparticles with sizes in the range of ∼300 to ∼1000 nm, whereas the SBA15-cal material consisted of irregularly shaped and micrometer sized particles ∼1000 nm long having typical diameters of ∼500 nm. The degree of condensation within the silica framework was determined for MCM41-cal and SBA15-cal samples by using solidstate 29Si{1H} cross-polarization magic angle spinning (CP-MAS) NMR spectroscopy. The spectrum for MCM41-cal is shown in Figure 1G, which shows well-defined (SiO)2Si(OH)2 (Q2), (SiO)3-SiOH (Q3), and (SiO)4-Si (Q4) silicate species, appearing at −92, −100, and −110 ppm, respectively.

Ventricular Myocyte Isolation. Experiments were performed in 6- to 8-week old male Wistar rats. Ventricular myocytes were isolated according to previously described techniques.22 Briefly, the animals were euthanized using a guillotine. The hearts were rapidly removed and mounted for retrograde perfusion using the Langendorff method. The hearts were perfused at 36 to 37 °C and a constant flow of 8.0 mL·g heart−1·min−1, using the following cell isolation solution (in mmol/L): 130 NaCl, 5.4 KCl, 1.4 MgCl2, 0.75 CaCl2, 0.4 NaH2PO4, 5.0 HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer, 10 glucose, 20 taurine, and 10 creatine (pH 7.3). When the heart had stabilized, perfusion was continued for 4 min with Ca2+-free isolation solution containing 0.1 mmol/L EGTA (ethylene glycol tetraacetic acid), followed by perfusion with isolation solution containing 0.05 mmol/L Ca2+, 0.75 mg/mL type 1 collagenase, and 0.075 mg/mL type XIV protease for 6 min. Ventricle tissue was then 28

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measured during washout with normal Tyrode. Data were acquired and analyzed with Signal Averager software v 6.37 (Cambridge Electronic Design, Cambridge, UK). Ventricular Myocyte Viability. Separate experiments were performed to assess the viability of ventricular myocytes in NT and in NT + MCM41-cal and NT + SBA15-cal particles at a concentration of 200 μg/mL over a time period of 120 min at 35 to 36 °C. A hemocytometer was used to count the number of rod shaped cells (displaying normal morphology) and round shaped cells and express the results as a percentage. In Vivo Studies. In order to determine whether MCM41-cal could penetrate the sarcolemma membrane of intact ventricular myocytes, 2.0 mg/mL solution of MCM41-cal was injected into the inferior vena cava of male Wistar rats (3 months old). After euthanasia, the apexes of the left ventricles of rats were excised 10 min after the injection and processed for TEM as described above. MCM41-cal was chosen out of the two particles used in this study because MCM41-cal is more electron-dense and aggregates in larger numbers around ventricular myocytes when compared to SBA15-cal. Moreover, it has been shown that MCM41-cal is a nontoxic microparticle with other murine tissues.4 Statistics. Results were expressed as the mean ± standard error of the mean of n observations. Statistical comparisons were performed using either the independent samples t-test or one-way ANOVA followed by Bonferroni's corrected t-tests for multiple comparisons, as appropriate. P < 0.05 was considered significant. Morphometric analysis of MCM41-cal particles located in the cytoplasm of ventricular myocytes was done blindly by counting the number of particles per a randomly chosen cell. The result was presented as the mean ± SDEV (n = 6).

excised, minced, and gently shaken in collagenase-containing isolation solution supplemented with 1% bovine serum albumin. Cells were filtered at 4-min intervals and resuspended in isolation solution containing 0.75 mmol/L Ca2+. Transmission Electron Microscopy (TEM). Ventricular myocytes were incubated at 35 to 37 °C for 10 min in normal Tyrode solution containing (in mmol/L): 140 NaCl, 5.0 KCl, 1.0 MgCl2, 10 glucose, 5.0 HEPES, 1.8 CaCl2 (pH 7.4), and 200 μg/mL MCM41-cal or SBA15-cal. The suspensions were centrifuged at 800g for 1 min. The supernatants were removed, and the pellets were resuspended in 0.1 M phosphate buffered saline (PBS). The tissue was fixed in a modified McDowell and Trump fixative for 1 h at 25 °C. After rinsing with PBS, the cells were postfixed with 1% osmium tetroxide for 1 h and washed in dH2O. The cells were dehydrated in a series of graded ethyl alcohol and propylene oxide before infiltration with resin. The cells were then embedded in Agar 100 epoxy resin and polymerized for 24 h at 65 °C. Blocks were trimmed, and semithin and utrathin sections were cut with Reichert Ultracut microtome. Semithin sections (0.5 μm thickness) were stained with 1% aqueous toluidine blue on glass slides. Ultrathin sections (95 nm) placed on 200 mesh copper grids were contrasted with uranyl acetate and lead citrate. The grids were examined and photographed using a Philips CM10 TEM instrument. Scanning Electron Microscopy (SEM). Ventricular myocytes were fixed with a Karnovsky fixative (2% paraformaldehyde and 2.5% glutaraldehyde) for 30 min at 25 °C, then washed three times with 0.1 M phosphate buffer at pH 7.2, and then post-fixed with chilled 1% osmium tetroxide for 1 h. Cells were then washed with distilled water three times for 5 min, then dehydrated with an ascending series of ethanol from 30% to 100% for 15 min each. The cells were subjected to critical point drying (Polaron CPD, Sussex, England) and later mounted on carbon tabbed 0.5 mm aluminum stubs. The stubs were then fixed in the Polaron Sputter Coater Vacuum chamber (Sussex, England) and were spattered with a Au/Pd target for 6 min at 20 mA current. Samples were examined with a SEM Philip-XL30, and pictures were taken at different magnifications. Ventricular Myocyte Shortening. Ventricular myocytes were allowed to settle on the glass bottom of a Perspex chamber mounted on the stage of an inverted Axiovert 35 microscope (Zeiss, Göttingen, Germany). Cells were superfused with normal Tyrode at 3−5 mL·min−1. Unloaded myocyte shortening was recorded using a VED-114 video edge detection system (Crystal Biotech, Northborough, MA, USA). Resting cell length, time to peak (TPK) shortening, time to half (THALF) relaxation, and amplitude of shortening (expressed as a percentage of resting cell length) were measured in field stimulated (1 Hz) myocytes maintained at 35 to 36 °C. Shortening was measured in normal Tyrode after 10 min of superfusion with normal Tyrode containing 200 μg/mL MCM41-cal or SBA15-cal. Shortening was also measured during washout with normal Tyrode. Data were acquired and analyzed with Signal Averager software v 6.37 (Cambridge Electronic Design, Cambridge, UK). Measuring Intracellular Ca2+. Ventricular myocytes were loaded with the fluorescent indicator Fura 2-AM (F-1221) as previously described.22 In brief, 6.25 μL of 1.0 mM Fura 2-AM solution (in dimethylsulfoxide) was added to 2.5 mL of cell suspension (final concentration, 2.5 μM). Myocytes were shaken gently at 24 °C for 10 min. After loading, the cells were centrifuged, washed with normal Tyrode, and left for 30 min to ensure complete hydrolysis of the intracellular ester. The myocytes were then alternately illuminated by 340 and 380 nm light, using a monochromator (Cairn Research, Faversham, UK) which changed the excitation wavelength every 2 ms. The resulting fluorescence emission at 510 nm was recorded; the ratio of the emitted fluorescence at the two excitation wavelengths 340 and 380 nm was calculated to provide an index of intracellular [Ca2+]. Resting Fura 2-AM ratio, time to peak (TPK) Ca2+ transient, time to half (THALF) decay of the Ca2+ transient, and the amplitude of the Ca2+ transient were measured in field stimulated (1 Hz) myocytes maintained at 35 to 36 °C. Ca2+ transients were measured in normal Tyrode after 10 min of superfusion with normal Tyrode containing 200 μg/mL MCM41-cal or SBA15-cal. Ca2+ transients were also

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RESULTS Transmission Electron Microscopy (TEM). The ability of MCM41-cal and SBA15-cal to penetrate the sarcolemma of isolated ventricular cardiomyocytes was investigated by TEM (Figure 2). MCM41-cal aggregated on the surface of isolated myocytes (Figure 2c) and eventually penetrated the plasma membrane after 10 min of incubation at 37 °C (Figure 2e). The cytoplasmic organelle most commonly associated with MCM41-cal was the mitochondrion (Figure 2e). Only a small proportion of the MCM41-cal that aggregated on the periphery of isolated cardiomyocytes actually entered the cell. The particles appeared more round in shape compared to their extracellular shape (Figure 2a) and settled around the mitochondria as soon as they entered the cell (Figure 2e). The SBA15-cal particles were less conspicuous and more amorphous compared to the MCM41-cal. In contrast to MCM41-cal which appeared in solitude, SBA15-cal appeared as fragmented particles around isolated myocytes (Figure 2d). However, SBA15-cal particles also attained a more rounded shape as soon as they entered the cell, where they attached to the mitochondrial membrane (Figure 2f). Similar to the pattern of distribution of MCM41-cal, only a small proportion of the particles entered the cell compared to the number of fragmented particles located around the outer surface of the cell. In order to determine whether the ability of particles to enter isolated ventricular myocytes was due to an injury of the plasma membrane following isolation and incubation, experimental animals were injected with 0.75 mL of 2.0 mg/mL 0.9% NaCl/ 5% glucose solution containing MCM41-cal. MCM41-cal (5.0 ± 0.02 particles per cell, n = 6) was capable of traversing the endothelial membrane of cardiac capillaries and eventually crossing the perivascular region before finally entering intact ventricular myocytes. Having gained entry into the cell, the particles migrated toward mitochondrial membranes of intact 29

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Figure 2. TEM micrographs showing (a) a group of MCM41-cal microparticles (arrow) as retrieved from the incubating solution; (b) a group SBA15-cal microparticles (arrow) as retrieved from the incubating solution; (c) MCM41-cal microparticles in the vicinity of isolated ventricular myocytes (arrow) incubated at 37 °C for 10 min (particles are found among cytoplasmic organelles, mainly the mitochondria); (d) fragments of SBA15-cal (arrowhead) around isolated ventricular myocytes incubated at 37 °C for 10 min (particles were found among cytoplasmic organelles, mainly the mitochondria); (e) MCM41-cal microparticles (arrow) inside isolated ventricular myocytes incubated at 37 °C for 10 min (note the MCM41-cal around mitochondrial membranes); and (f) SBA15-cal fragments (arrowhead) around mitochondria. SBA15-cal fragments (arrow) have attached to the outer membrane of the mitochondria of an isolated ventricular myocyte incubated at 37 °C for 10 min. m = mitochondrion. Scale bar = 1 μm.

Figure 3. TEM micrographs showing different stages in the journey of MCM41-cal from the perivascular region to its final destination, the mitochondrial membrane in an intact ventricular myocyte, after intravenous injection of 2.0 mg/mL of MCM41-cal. (A,E) MCM41-cal in the perivascular area (arrowhead) and the membrane of mitochondria in the interior of the cell (arrow); (B,F) MCM41-cal between stacks of mitochondria; (C,D) many MCM41-cal particles were consistently located on the membrane of mitochondria. m = mitochondrion; c = capillary. Scale bar = 1 μm.

normal Tyrode + MCM41-cal (200 μg/mL for 2, 5, and 10 min), and washout for 10 min with normal Tyrode (Figure 5). Resting cell length (RCL, in μm) was not significantly (p > 0.05) altered during the superperfusion of myocytes with MCM41-cal. Time-to-peak (TPK) shortening (in ms) and time-to-half (THALF) relaxation of shortening (in ms) were also not significantly (p > 0.05) altered during superperfusion with MCM41-cal. Interestingly, the amplitude of shortening (percent resting cell length, % RCL) was modestly and progressively reduced during the superperfusion of myocytes with MCM41-cal for 2, 5, and 10 min; however, the changes did not reach statistical significance (p > 0.05). In normal Tyrode without particles, the amplitude of shortening was 6.51 ± 1.00% (n = 14 cells), compared to 4.51 ± 0.72% (n = 13 cells) after 10 min of superperfusion with normal Tyrode + MCM41-cal, a reduction in amplitude of 69.3%. After incubation at a concentration of 50 μg/mL MCM41-cal for 10 min, the reduction in amplitude was 69.5% (data not shown). There was no significant (p > 0.05) recovery of

ventricular myocytes (Figure 3A−F). Thus, in both in vitro and in vivo studies, MCM41-cal consistently settled around and attached to mitochondrial membrane. Scanning Electron Microscopy (SEM). SEM was done to examine the relationship of MCM41-cal and SBA15-cal to the surface of ventricular myocytes. The results showed that MCM41-cal settled on the surface of the plasma membrane of the ventricular cardiac muscle cell after incubation at 37 °C for 10 min (Figure 4c). SBA15-cal, however, settled on the surface of ventricular myocytes firmly attached to the plasma membrane (Figure 4d). Effects of Particles on Myocyte Shortening. Shortening was measured in electrically stimulated (1 Hz) ventricular myocytes maintained at 35 to 36 °C in normal Tyrode (NT), 30

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shortening during the 10-min washout with normal Tyrode (Figure 5). The same trend was observed during the superperfusion of myocytes with SBA15-cal (200 μg/mL, Figure 6). Resting cell length, time-to-peak (TPK) shortening, and time-to-half (THALF) relaxation were not significantly (p > 0.05) altered during the superperfusion of myocytes with SBA15-cal for 2, 5, and 10 min. The amplitude of shortening was modestly and progressively reduced during the superperfusion of myocytes with SBA15-cal for 2, 5, and 10 min; however, the differences did not reach statistical significance (p > 0.05). In normal Tyrode without SBA15-cal, the amplitude of shortening was 4.98 ± 0.53% (n = 15 cells) compared to 3.63 ± 0.33% (n = 13 cells) after 10 min of superperfusion with normal Tyrode + SBA15-cal, a reduction in amplitude of 72.9%. At a concentration of 50 μg/mL SBA15-cal for 10 min, the reduction in amplitude was 52.3% (data not shown). There was no significant (p > 0.05) recovery of shortening during the 10-min washout with normal Tyrode (Figure 6). Thus, the incubation of myocytes with MCM41-cal or SBA15-cal had no significant effect on the amplitude and the time course of shortening. Effects of Particles on Intracellular Ca2+. Intracellular [Ca2+] was measured in electrically stimulated (1 Hz) ventricular myocytes maintained at 35 to 36 °C in normal Tyrode (NT), with normal Tyrode + MCM41-cal (200 μg/mL for 2, 5, and 10 min), and then washout with normal Tyrode for 10 min (Figure 7). Resting Fura 2-AM ratio (340/380 nm) was

Figure 4. Scanning electron microscopy micrographs showing: (a) an aggregate of MCM41-cal (arrow) in the vicinity of a cardiomyocyte (arrowhead) and (b) an aggregate of SBA15-cal (arrow) near a ventricular myocyte (arrowhead). (c) MCM41-cal (arrow) has now settled inside a ventricular cardiac muscle cell (arrowhead) after incubation at 37 °C for 10 min. (d) SBA15-cal (arrow) lying on the surface of isolated ventricular myocytes (arrowhead) incubated at 37 °C for 10 min. Scale bar = a − b = 50 μm; c = (20 μm); d = 10 μm.

Figure 5. Effects of MCM41-cal on ventricular myocyte shortening. Shortening was measured in electrically stimulated (1 Hz) myocytes superfused at 35 to 36 °C with normal Tyrode, normal Tyrode plus MCM41-cal (200 μg/mL for 2, 5, and 10 min), and washout with normal Tyrode for 10 min. Data are the mean ± SEM, n = 12 to 14 cells. RCL, resting cell length; TPK, time-to-peak; THALF, time-to-half; NT, normal Tyrode. 31

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Figure 6. Effects of SBA15-cal on ventricular myocyte shortening. Shortening was measured in electrically stimulated (1 Hz) myocytes superfused at 35 to 36 °C with normal Tyrode, normal Tyrode plus SBA15-cal (200 μg/mL for 2, 5, and 10 min), and washout with normal Tyrode for 10 min. Data are the mean ± SEM, n = 12 to 15 cells. RCL, resting cell length; TPK, time-to-peak; THALF, time-to-half; NT, normal Tyrode.

Effects of Particles on the Viability of Myocytes. At time zero, the viability of rod shaped myocytes in NT was 53.87 ± 1.57% (n = 5) (Figure 9). Myocytes were then incubated in NT or NT + MCM41-cal or NT + SBA15-cal at a concentration of 200 μg/mL over a time period of 120 min at 35 to 36 °C. At 120 min, the viability of myocytes in NT was 31.00 ± 1.05%, in NT + MCM41-cal was 26.60 ± 1.60%, and in NT + SBA15-cal was 22.80 ± 3.12%; these differences were not significant (p > 0.05). Collectively, the results show that 10-min incubation of myocytes with MCM41-cal (but not with SBA15-cal) significantly reduced the amplitude of Ca2+ transient.

not significantly (p > 0.05) altered during superperfusion of myocytes with MCM41-cal. Time-to-peak (TPK) of the Ca2+ transient (in ms) and time-to-half (THALF) decay of Ca2+ were not significantly (p > 0.05) altered during superperfusion with MCM41-cal. Interestingly, the amplitude of the Ca2+ transient (Fura 2-AM ratio) was significantly reduced during the superperfusion of myocytes for 10 min with MCM41-cal. In normal Tyrode without MCM41-cal, the amplitude of the Ca2+ transient was 0.32 ± 0.03 (n = 14 cells), and was significantly (p < 0.05) reduced to 0.22 ± 0.03 (n = 11 cells) after 10 min of superperfusion with normal Tyrode + MCM41-cal. There was no significant (p > 0.05) recovery of the amplitude of Ca2+ transient during the 10-min washout with normal Tyrode (Figure 7). The same trend was observed during the superperfusion of myocytes with normal Tyrode containing 200 μg/mL SBA15cal (Figure 8). Resting Fura 2-AM ratio, time-to-peak (TPK) of the Ca2+ transient, and time-to-half (THALF) decay of the Ca2+ transient were not significantly (p > 0.05) altered during the superperfusion of myocytes with SBA15-cal for 2, 5, or 10 min. The amplitude of the Ca2+ transient was modestly reduced during the superperfusion of myocytes with SBA15-cal for 10 min; however, the change did not reach significance (p > 0.05). In normal Tyrode without SBA15-cal, the amplitude of the Ca2+ transient was 0.24 ± 0.03 (n = 11 cells), compared to 0.18 ± 0.02 (n = 11 cells) after 10 min of superperfusion with normal Tyrode + SBA15-cal. There was no significant (p > 0.05) recovery of the Ca2+ transient during the 10-min washout with normal Tyrode.

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DISCUSSION This study investigated the in vivo and in vitro biocompatibilities of two forms of calcined mesoporous silica particles, MCM41cal and SBA15-cal, in rat ventricular myocytes. Physical Characteristics of MCM41-cal and SBA15-cal. Compared to MCM-41 (regular spherical and oval-shaped), SBA15 (irregular rod-shaped) exhibits greater lattice spacing, bigger Barrett−Joyner−Halenda (BJH) pore diameters, and smaller Brunauer−Emmett−Teller (BET) surface areas. These differences may play a role in the way they react with cytoplasmic orgenelles or their ability to deliver bioactive substances to cells. TEM studies showed that MCM41-cal is more electron dense compared to SBA15-cal and that MCM41cal can also maintain its structure after incubation. In contrast, SBA15-cal disintegrated easily and became fragmented after incubation. The tendency of SBA15-cal to disintegrate may be 32

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Figure 7. Effects of MCM41-cal on intracellular Ca2+. Ca2+ transients were measured in electrically stimulated (1 Hz) myocytes superfused at 35 to 36 °C with normal Tyrode, normal Tyrode plus MCM41-cal (200 μg/mL for 2, 5, and 10 min), and washout with normal Tyrode for 10 min. Data are the mean ± SEM, n = 11 to 14 cells. TPK, time-to-peak; THALF, time-to-half; NT, normal Tyrode. The horizontal lines above the bars in the amplitude of the Ca2+ transient (Fura-2 ratio) panel represent p < 0.05.

protons and become negatively charged silanolate −SiO− ions, even at neutral pH.25,26 Most importantly, the two materials, MCM41-cal and SBA15-cal, have surface structural differences, i.e., MCM41-cal has a rather smooth surface, while SBA15-cal has corrugated and microporous surface structures.27 This leads to differences in their surface silanol density, which in turn might lead to different interactions to various cellular parts. Furthermore, this structural difference as well as their differences in shape (MCM41-cal is round or oval shaped, whereas SBA15-cal is rod shaped; see Figure 1) itself might cause the observed differences in the materials’ ability to interact with cellular parts. In Vivo Study. MCM41-cal was chosen for the in vivo study because the in vitro study showed that the number of particles that entered the inner core of ventricular myocytes was significantly higher in MCM41-cal compared to that of SBA15cal, indicating that MCM41-cal can get into the cells more easily. The ultrastructure of myocytes was well preserved in the presence of either particle, indicating that these particles are easily taken up by myocytes. Although the mechanism by which they enter ventricular myocytes was not investigated, it is likely that they have entered the cells by endocytosis. The compatibility of MCM41-cal and SBA15-cal with ventricular myocyte structure could be attributed to the refined methods of particle preparation, i.e., lacking residual surfactant molecules. MCM-41 and SBA-15 with surfactants extracted by solvent

due to other physical properties including its rough surface. This might indicate that MCM41-cal is superior to SBA15-cal in the delivery of drugs or bioactive agents. Bioavailability and Biocompatibility of MCM41-cal and SBA15-cal. Both MCM41-cal and SBA15-cal were reasonably bioavailable and biocompatible with ventricular myocytes since MCM41-cal and SBA15-cal were found inside the cell after a relatively short incubation period of 10 min. However, it is worth noting that only a small proportion of the fragmented SBA15-cal particles actually entered the cells, compared to MCM41-cal. The ability of MCM41-cal and SBA15-cal to enter cells might be due to the apparent high affinity of the particles toward cellular membranes as demonstrated by their unique chemical (e.g., surface charges) and physical properties. Both MCM41cal and SBA15-cal are known to possess surface silanol (-Si− OH) groups, as also confirmed here by 29Si solid state NMR spectroscopy (please see above and Figure 1G).23,24 Owing to their hydrophilic nature, these silanol groups generally make these particles mobile in aqueous media or cellular media. Furthermore, because of their ability to form hydrogen bonds as well as participate in electrostatic interaction, these silanol groups of mesoporous silica materials make the particles interact with cellular parts or organelles. Their ability to participate in electrostatic interaction stems from the fact that silanols are weakly acidic, and thus, some of them lose their 33

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Figure 8. Effects of SBA15-cal on intracellular Ca2+. Ca2+ transients were measured in electrically stimulated (1 Hz) myocytes superfused at 35 to 36 °C with normal Tyrode, normal Tyrode plus SBA15-cal (200 μg/mL for 2, 5, and 10 min), and washout with normal Tyrode for 10 min. Data are the mean ± SEM, n = 11 cells. TPK, time-to-peak; THALF, time-to-half; NT, normal Tyrode.

MCM41-cal on intracellular Ca2+ was more pronounced than the effects of SBA15-cal. Incubation of the myocytes with MCM41-cal (but not with SBA15-cal) significantly reduced the amplitude of the Ca2+ transient. Superperfusion of ventricular myocytes with MCM41-cal or SBA15-cal for 10 min caused a modest, yet statistically insignificant, reduction in the amplitude of shortening. Interestingly, the effects of the particles on shortening were not reversed during the 10-min washout. The negative inotropic effects of the particles may be attributed to a variety of causes, including mechanical abrasive damage interfering with membrane transport systems. The viability of myocytes, expressed as the percentage of rod shaped compared to round shaped myocytes, over a 120 min period was not significantly different in cells incubated in NT, compared to that in NT + MCM41-cal or NT + SBA15-cal particles suggesting that incubation of cells with particles per se had no detrimental effects on the viability of cells. During the process of excitation−contraction, coupling the arrival of an action potential leads to depolarization of the cell membrane and opening of the voltage-gated L-type Ca2+ channels. There is a small entry of Ca2+ via L-type Ca2+ channels, which then triggers a larger release of Ca2+ from the sarcoplasmic reticulum. As a result, there is a transient rise of intracellular Ca2+ (Ca2+ transient). The Ca2+ binds to troponin C, thereby initiating and regulating the process of contraction. Interestingly, 10-min superperfusion with MCM41-cal (though not SBA15-cal) significantly reduced the amplitude of the Ca2+ transient, suggesting altered mechanisms of Ca2+ transport might partly underlie the negative inotropic effects of these particles.

Figure 9. Effects of MCM41-cal on myocyte viability. A hemocytometer was used to count the number of rod shaped cells (displaying normal morphology) and round shaped cells and express the results as a percentage. Ventricular myocytes were incubated in either NT, NT + MCM41-cal, or NT + SBA15-cal at a concentration of 200 μg/mL over a time period of 120 min at 35 to 36 °C. Data are the mean ± SEM, cells from 5 hearts.

washing were not biocompatible with murine tissues or hematopoietic cells.4,13 These findings emphasize the impact of the synthetic procedures on particle toxicity. Effect of MCM41-cal and SBA15-cal on Ventricular Myocyte Physiology. Incubation of ventricular myocytes with MCM41-cal or SBA15-cal had no significant effect on the amplitude and the time course of shortening. The effect of 34

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The reduction in the amplitude of shortening and Ca2+ transient observed in ventricular myocytes after incubation with MCM41-cal could be due to several factors. The electrical charge on the particles may affect the charge between the inner and outer parts of the plasma membranes, thereby affecting the resting potential and the ability of myocytes to respond to the electrical stimulation. This possibility is supported by the scanning electron microscopy, which showed MCM41-cal and SBA15-cal attached to the plasma membranes of ventricular myocytes after 10 min of incubation. Moreover, MCM41-cal and SBA15-cal had an apparent affinity for the mitochondrial membrane. Therefore, in metabolically active cells, such as cardiomyocytes, an impaired oxidative phosphorylation (inadequate cellular bioenergetics) leading to a reduction in effective shortening is not surprising. Clinical Significance of MCM41-cal and SBA15-cal. The present study showed that these particles have affinity for ventricular muscle mitochondria and may thus play a role in the modulation of mitochondrial function. Attachment of these particles to the mitochondrial membrane might affect enzymes that regulate the electron transport chain. In addition, these microparticles can also be used to carry specific drugs and or bioactive agents to cardiac muscles or the mitochondria in particular. Mitochondriopathy contributes to the etiopathogenesis of several diseases. Drugs carried by MCM41-cal and SBA15-cal may also target areas of myocardial infarction. The possibility of large particle aggregates blocking small capillaries is realistic. This physical property of the particles, however, depends largely on concentration, dispensing solution, dosing, schedule of administration, and perfusion time. Certainly, these and many other variables need to be considered before a safe and effective clinical regimen could be recommended for clinical use. In conclusion, MCM41-cal and SBA15-cal are reasonably biocompatible with rat ventricular myocytes. These particles, especially MCM41-cal, rapidly penetrate ventricular myocytes even after a relatively short incubation or i.v. injection. Particles that managed to enter the cells settle around the mitochondrial membrane. This proximity to the mitochondria could be responsible for the observed reduction in the amplitude of shortening and the Ca2+ transient in ventricular myocytes. Optimal use of these particles may have an important clinical role in the management of heart diseases.

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Brunauer, Emmett, and Teller; CP MAS, cross-polarization magic-angle spinning; NMR, nuclear magnetic resonance.

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AUTHOR INFORMATION

Corresponding Author

*(E.A.) Tel: 00971-3-973496. Fax+971-3-973496. E-mail: [email protected]. (F.C.H.) Tel: 00971-3-973536. Fax +971-3-7137536. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

ABBREVIATIONS

MCM, mobil composition of matter; SBA, Santa Barbara amorphous; HL, human leukocyte; CTAB, cetyltrimethylammonium bromide; TEOS, tetraethyl orthosilicate; TEM, transmission electron microscopy; SEM, scanning electron microscopy; PBS, phosphate buffered saline; VED, video edge detection system; TPK, time to peak; THALF, time to half; RCL, resting cell length; Fura 2-AM, Fura 2-acetoxymethyl; NT, normal Tyrode; BJH, Barrett−Joyner−Halender; BET, 35

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