Influence of Silica Nanoparticle Density and Flow Conditions on

Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Influence of Silica Nanoparticle Density and Flow Conditions on Sedimentation, Cell Uptake, and Cytotoxicity Mostafa Yazdimamaghani,†,‡ Zachary B. Barber,§ Seyyed Pouya Hadipour Moghaddam,†,‡ and Hamidreza Ghandehari*,†,‡,§ †

Utah Center for Nanomedicine, Nano Institute of Utah, ‡Department of Pharmaceutics and Pharmaceutical Chemistry, Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112, United States

§

ABSTRACT: Careful evaluation of the toxicological response of engineered nanomaterials (ENMs) as a function of physicochemical properties can aid in the design of safe platforms for biomedical applications including drug delivery. Typically, in vitro ENM cytotoxicity assessments are performed under conventional static cell culture conditions. However, such conditions do not take into account the sedimentation rate of ENMs. Herein, we synthesized four types of similar size silica nanoparticles (SNPs) with modified surface roughness, charge, and density and characterized their cytotoxicity under static and dynamic conditions. Influence of particle density on sedimentation and diffusion velocities were studied by comparing solid dense silica nanoparticles of approximately 350 nm in diameter with hollow rattle shape particles of similar size. Surface roughness and charge had negligible impact on sedimentation and diffusion velocities. Lower cellular uptake and toxicity was observed by rattle particles and under dynamic conditions. Dosimetry of ENMs are primarily reported by particle concentration, assuming homogeneous distribution of nanoparticles in cell culture media. However, under static conditions, nanoparticles tend to sediment at a higher rate due to gravitational forces and hence increase effective doses of nanoparticles exposed to cells. By introducing shear flow to SNP suspensions, we reduced sedimentation and nonhomogeneous particle distribution. These results have implications for design of in vitro cytotoxicity assessment of ENMs and suggest that among other factors, sedimentation of nanoparticles in toxicity assessment should be carefully considered. KEYWORDS: engineered nanomaterials, silica nanoparticles, nanotoxicity, sedimentation, density

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INTRODUCTION Advances in the synthesis and characterization of engineered nanomaterials (ENMs) are not matched by careful assessment of their effects on environmental health and safety. Intended and unintended occupational and consumer exposures to ENMs pose potential risk for nanomaterials, for which there is limited toxicological knowledge.1−6 It is known that physicochemical factors such as size, size distribution, shape, porosity, surface functionality, surface area, charge, and composition influence cellular uptake and toxicity of ENMs.7−14 Little is known however about the influence of ENM density and flow conditions on corresponding sedimentation, cellular uptake, and toxicity. The majority of in vitro ENM cytotoxicity assessments are performed under conventional static cell culture conditions, and dosimetry is reported by particle mass, number, or surface area per unit © XXXX American Chemical Society

volume of media. Under static conditions, nanoparticles tend to sediment due to gravitational forces, which in turn can change the effective dosimetry (Scheme 1).15 Sedimentation and diffusion mass transport phenomena of ENMs, governed by sedimentation and diffusion coefficients, are dependent on the diameter and density of particles as well as flow rate and viscosity.16−20 It has been reported that for particles larger than 100 nm, mass transport is predominantly governed by sedimentation rather than diffusion,17,18 pointing to the need for a more-detailed understanding of the influence of sedimentation, flow, and density of particles on toxic response. Received: Revised: Accepted: Published: A

February 26, 2018 April 8, 2018 May 2, 2018 May 2, 2018 DOI: 10.1021/acs.molpharmaceut.8b00213 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Scheme 1. (A) Prepared Spherical SNPs of Similar Size with Different Surface Roughness, Charges, and Densities; (B) Static Conditions in Conventional Cell Culture Plates; (C) Dynamic Conditions on Tilting Shakera

a (A) Spherical particles of similar size were prepared with different surface roughness, charges, and densities while controlling for other differences between particle types to examine the effect of altered physicochemical properties on cell uptake, sedimentation, and cytotoxicity. Schematics of nanotoxicity studies based on conventional static (B) and dynamic cell culture exposed to flow on tilting shaker (C). Sedimentation of solid dense Stöber SNPs with nonhomogeneous distribution of nanoparticles is compared with hollow rattle structured SNPs with homogeneous distribution. Both high and low density particles exposed to flow show less sedimentation and more-homogeneous distribution.

homogeneous distribution of nanoparticles and led to increased percentage of viable cells compared to static conditions.15 One challenge with the use of microfluidic systems with the polydimethylsiloxane (PDMS) structure is potential adsorption of nanoparticles to PDMS, which in turn can alter the effective particle concentrations. While microfluidics can be useful in mimicking physiological flow, they are complex and expensive. An alternative method for investigating the effect of sedimentation on particle uptake is the use of inverted cell culture conditions. Cho et al. reported the uptake of gold nanoparticles with different sizes, shapes, and densities of cells at the bottom of a conventional culture and under inverted cell culture configurations.29 A challenge with this method is that cells positioned in the upright configuration are exposed to both sedimentation and diffusion of nanoparticles from media, whereas cells in the inverted configuration are accessible only by the nanoparticles’ diffusion transport. In addition, sedimentation of nanoparticles below the cells and on the upper side of the coverslip in the inverted configuration can eventually influence the effective concentration of particles. Further, cells can migrate from an inverted section to the bottom of a well-plate, in which case, not all cells are exposed to the same dosage of nanoparticles. To address these issues, we have presented a simple, rapid, and cost-effective method of introducing dynamic flow conditions utilizing a tilting shaker to evaluate the sedimentation effect on in vitro toxicity. This method is simple, inexpensive, and reproducible. To further optimize the system,

Compared to conventional static cell culture studies, dynamic conditions provide the opportunity to prevent nonhomogeneous distribution of nanoparticles and a more-accurate dosimetry. In addition, the resulting shear stress and increased accessibility of the nutrients to cells by agitation can influence cellular response to ENMs. In most cases, a typical in vitro cellular assay is utilized to assess the cytotoxicity of ENMs. Although conventional in vitro cellular assays can be effectively used for safety evaluation of small molecule substances, they cannot ensure meaningful in vitro cytotoxicity evaluation of colloidal suspensions of ENMs due to ENMs’ interactions with media, agglomeration, transport, and sedimentation.21−25 There is a significant need for developing fast, affordable, and reliable nanotoxicity screening methodologies.26 Demokritou et al. proposed a multistep in vitro dosimetric experimental/computational methodology to quantify the delivered dose of ENMs to cells in a typical cellular assay. This protocol takes into account the “particokinetics” and includes three parts of ENMs’ preparation, characterization, and volumetric centrifugation method in vitro sedimentation, diffusion, and dosimetry (VCM−ISDD computational transport modeling).27,28 Other reports have also focused on studies assessing the influence of ENM density and the sedimentation effect on cell uptake and toxicity.15,29,30 With the use of microfluidic devices to generate a concentration gradient of nanoparticles on cultured cells, the toxicity of quantum dots (QD) was evaluated.15 Avoiding the gravitational settlement of QDs by flow in microfluidics chips provided a B

DOI: 10.1021/acs.molpharmaceut.8b00213 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics Scheme 2. Schematic of Silica Nanorattle Synthesisa

a

Silica nanorattles (RL) were synthesized via a layer-by-layer modified Stöber method. Particle cores were synthesized using a typical Stöber reaction, with the addition of FITC and APTES. The middle layer of RL particles was synthesized by co-condensing TEOS with TSD into a less-dense organic layer. A dense shell layer of solid silica was then added around the middle layer. The organic middle layer was then selectively etched out using concentrated hydrofluoric acid. Etching left the particles with a solid core inside a hollow interior, surrounded by a porous shell.

one could add shear measurement devices to elaborate the influence of tilting speed on the sedimentation effect. In addition, there is a limitation with the well plate types used on tilting shakers due to capillary forces in well plates with more than 12 wells in each plate. Herein, we used silica nanoparticles (SNPs) as model ENMs to investigate the influence of nanoparticle density and flow conditions on sedimentation, cell uptake, and cytotoxicity. Due to their chemical stability, ease of control over size, geometry, surface modification, and economic affordability, SNPs provide a suitable platform for this purpose.31,32 As shown in Scheme 1, four different types of SNPs were synthesized with altered surface roughness, charge, and density. Size, morphology, charge, hydrodynamic diameter, density, and surface area of the nanoparticles were characterized. Positively charged hollow structured rattle shape SNPs were synthesized using a selective etching procedure. Solid nonporous Stö ber SNPs with approximately 350 nm in diameter were synthesized and characterized. Surface roughness and charge were modified to obtain the same surface properties as those of low density hollow structured SNPs. To evaluate the effect of density and flow on the toxicity and cellular uptake of SNPs, Raw264.7 macrophages were treated with particles under conventional static cell culture conditions, and dynamic conditions were facilitated by a tilting shaker.

Fisher Scientific (Eugene, OR, U.S.A.). All chemicals were used as received without further purification. Synthesis of Silica Nanoparticles. Nonporous SNPs with a defined shape and size were prepared as reported previously.11 In brief, spherical nonporous SNPs of 357 ± 22.8 (SNE) were synthesized by a modified Stöber method.11,33 Silica nanorattles (RL) were synthesized as shown in Scheme 2 via a two-step selective etching process reported previously.34 Three solutions were prepared. Solution A contained 18 mL of absolute ethanol and 2 mL of tetraethyl orthosilicate (TEOS). Solution B contained 60 mL of absolute ethanol and 20 mL of ammonium hydroxide (28% aq). Solution C contained 8 mL of absolute ethanol and 200 μL of N-[3-(trimethoxysilyl) propyl]ethylenediamine (TSD). Solution B was placed in an Erlenmeyer flask and stirred at 400 rpm at room temperature. Solution A (5 mL) was added dropwise at a rate of 1 mL/min to solution B and allowed to stir for 10 min. Solution C and 8 mL of solution A were then added synchronously at a rate of 1 mL/min to the flask. Remaining solution A was then added all at once, and the solutions were allowed to stir for 3 h. Particles were then washed with DI H2O three times. In the second step, prepared particles were etched using HF. Particles suspended in DI H2O (1 mL) at a concentration of 9 mg/mL were placed in a 1.5 mL microcentrifuge tube. Then, 15 μL of 10% HF (aq) was added to the particles. At a setting of 7/10 of maximum vortex rate, particles were vortexed for 5 min to facilitate etching. Scheme 2 further illustrates the steps for the synthesis of RL SNPs. Surface-etched Stöber particles (SE) were synthesized by etching prefabricated Stöber particles (SNE) under precisely the same concentration and conditions as the RL SNPs. Amineterminated surface-etched Stö ber particles (SE+) were synthesized using a portion of the SE particles. SE particles suspended in absolute ethanol (20 mL) at a concentration of approximately 10 mg/mL were added to a round-bottom flask along with 80 mL of absolute ethanol. The solution was stirred at 500 rpm under anhydrous conditions, obtained using an N2filled balloon. (3-Aminopropyl)triethoxysilane (APTES, 4.2 × 10−3 M) was added. The solution was stirred for 24 h and then washed using absolute ethanol. The particles were stored at 4 °C to prevent degradation. All different types of prepared SNPs were washed three times by centrifugation at 27 000 RCF for 15 min, and subsequently, the supernatant was replaced with ethanol. Synthesized SNPs were then stored in ethanol for future experiments. Characterization of Silica Nanoparticles. Transmission electron microscopy (TEM) with a JEOL JEM 1400 microscope (JEOL Ltd., Tokyo, Japan) operating at 120 kV and

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MATERIALS AND METHODS Materials. N-[3-(Trimethoxysilyl) propyl]ethylenediamine (TSD), tetraethyl orthosilicate (TEOS, 98%), Triton X-100, (3aminopropyl)triethoxysilane (APTES), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, U.S.A.). Ammonium hydroxide (NH4OH, 28−30% as NH3) and hydrofluoric acid (HF 48%) were obtained from EMD Millipore Corporation (Billerica, MA, U.S.A.). Absolute ethanol (200 proof) was purchased from Decon Laboratories, Inc. (King of Prussia, PA, U.S.A.). Trypan blue stain 0.4% was received from Invitrogen (Carlsbad, CA, U.S.A.). RAW 264.7 macrophages (ATCC TIB-71) were acquired from the American Type Culture Collection (ATCC, Manassas, VA, U.S.A.). Phosphate buffered saline (PBS) Biotechnology grade tablets, hydrochloric acid (ACS-grade BDH, 36.5−38.0%), and Roswell Park Memorial Institute (RPMI) media were received from VWR (Radnor, PA, U.S.A.). Cell counting kit-8 (CCK-8) cytotoxicity assay was received from Dojindo (Rockville, MD, U.S.A.). ActinRed 555 ReadyProbes reagent, Hoechst, lactate dehydrogenase (LDH) assay, LysoTracker Deep Red, and fluorescein isothiocyanate (FITC) were received from Thermo C


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PBS twice. RPMI media with 10% fetal bovine serum providing varying concentrations of 8 to 250 μg/mL of SNPs were incubated with cells up to 24 h. For control samples, fresh media was added without SNPs. Then, the media was aspirated, and the cells were washed twice with PBS. Cell viability was determined with a CCK-8 assay, and the membrane integrity was evaluated by the LDH Cytotoxicity Assay Kit according to the manufacturer’s protocol. Cell Uptake. The uptake of silica nanoparticles by RAW 264.7 macrophages was evaluated by TEM, inductive coupled plasma-mass spectrometry (ICP-MS), and flow cytometry. Cells with 200 000 cells per well density were seeded on 6 well plates covered by 1 × 1 cm ACLAR plastic sheets and incubated for 24 h at 37 °C in 5% CO2. Next, cells were washed and treated with the LC50 concentrations of different types of SNPs and incubated for 4 h under two different conditions of static and laminar flow. Then, cells were washed three times with PBS and fixed with 1 mL of fixing solution (2.5% glutaraldehyde + 1.0% paraformaldehyde). Cells were postfixed with osmium tetroxide (OsO4), en-bloc stained with uranyl acetate, dehydrated, and embedded in an epoxy resin. Lastly, ultrathin sections of stained cells utilizing a diamond knife were prepared, which were then stained sequentially with uranyl acetate and Reynold’s lead-citrate for imaging at room temperature by a JEOL JEM 1400 microscope at an accelerating voltage of 120 kV. ICP-MS was used to quantify SNP internalization and association with cells using an Agilent 7500ce inductive coupled plasma-mass spectrometer (Santa Clara, CA, U.S.A.). For ICP-MS measurements, cells were treated with SE+ and RL SNPs at the same concentration of 100 μg/mL and incubated for 24 h to evaluate cell uptake. Concentrations in ICP-MS were chosen in such a way to prevent toxicity and loss of SNPs by washing away dead cells containing particles. Control samples were prepared using media without particles. The cell number for each time point was determined for each particle type. Next, the cells were washed three times with PBS and collected to prepare cell pellets, after which they were transferred to a PTFE (Savillex) vial to allow liquid content to evaporate. Cells were digested and dried with 0.5 mL of trace metal grade HNO3 at 150 °C on the hot plate for 2 h. The oxidized product was dissolved in 2 mL of water and transferred into a PS auto sampler tube. A known standard Si reference was taken to generate the standard curve. An internal standard of Cs was added to each sample. An Agilent 7500ce ICP-MS instrument was used under operating conditions suitable for routine multielement analysis. All of the chemicals used were of trace metal grade. The number of SNP associations per cell was calculated via the silicon content, cell density, and pore volume of particles to provide a quantitative measurement of SNP association. In addition, the cellular association of FITC-doped particles was measured by flow cytometry, and for each specimen, a minimum of 10 000 events was recorded. Cell Morphology and Colocalization. To test the influence of the dynamic conditions on cell morphology changes, in the absence of SNPs, cells were seeded, incubated, and washed on a six-well plate, similar to the toxicity studies. ActinRed 555 ReadyProbes Reagent as a F-actin probe and Hoechst dye were used to stain cells according to the manufacturer’s protocol. Next, the staining solutions were aspirated, and the cells were washed three times with PBS to remove excess stains. Confocal laser scanning microscopy was employed, and images were acquired by an Olympus FluoView

scanning electron microscopy (SEM) with a FEI Quanta 650 FE-SEM (Hillsboro, OR, U.S.A.) operating at 20 kV were used to characterize the morphology and size of prepared nanoparticles. A Malvern Instruments Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.) was utilized to measure the hydrodynamic diameter and zeta potential of synthesized SNPs. The particle density was measured by packed cell volume (PCV) tube centrifugation. Nitrogen adsorption−desorption isotherm analysis was conducted at −196 °C on a Micrometrics ASAP 2020 (Norcross, GA, U.S.A.) for determining surface area and pore size. Theoretical sedimentation velocities were calculated from particle densities using the sedimentation velocity equation. Particle Density Measurement. The densities of silica nanoparticles were measured using centrifugation in packed cell volume (PCV) tubes.35 Pellet volumes were measured using PCV, and knowing the total mass of silica in each tube, the density was calculated. It was assumed that, during centrifugation, pellets formed in a manner corresponding to random close stacking; thus, a stacking factor of 0.634 was used in calculations. The density of the particles was calculated for both dry and wet states. Media absorption and protein corona formation and subsequent density changes comparing dry and wet conditions were taken into account for the hollow structured RL particles as well as adsorptions onto the surface of solid particles. The corresponding change in density in the wet state was calculated using eq 1, where a media density (ρmedia) of 1 g/mL and silica density (ρENM) of 2 g/mL were used, and the pellet mass and volume (MENM and Vpellet respectively) were obtained via particle centrifugation in PCV tubes. ⎡⎛ ⎞⎛ ⎞⎤ ρ M ρev = ρmedia + ⎢⎜⎜ ENM ⎟⎟⎜⎜1 − media ⎟⎟⎥ ⎢⎣⎝ VpelletSF ⎠⎝ ρENM ⎠⎥⎦

(1)

Cell Culture and Cytotoxicity Assay. The cell density was investigated by counting the cell number utilizing a Countess Automated Cell Counter (Thermo Fisher Scientific Corporation, Grand Island, NY, U.S.A.) and normalizing for the area in which the cells were cultured. Cells were seeded onto 6-well plates with 200 000 cells/well and allowed to grow, up to 3 days. Cell densities were determined for both static and dynamic conditions up to 3 days, in which each value was obtained by the mean of 6 counts. Cell proliferation was investigated by the Click-iT EdU microplate assay kit (Thermo Fisher Scientific) following manufacturer provided protocols. DNA synthesis was measured by incorporation of the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU) into DNA with EdU coupled to Oregon Green-azide labeling, after which HRP-conjugated anti-Oregon Green antibody and Amplex UltraRed would reveal EdU incorporation. The fluorescence was then measured at an excitation/emission wavelength of 490/585 nm utilizing a SpectraMax M2 (Molecular Devices, Sunnyvale, CA, U.S.A.) microplate reader. Measurements were conducted in triplicate. The cytotoxicity of the four different types of SNPs with varying surface roughness, charge, and density were tested on RAW 264.7 macrophages in the presence and absence of media suspension agitation. Cells were cultured in RPMI with 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. For the cytotoxicity assays, cells were seeded onto 6-well plates with 200 000 cells/well and allowed to attach to wells for 24 h. After 24 h, the media was aspirated, and the cells were washed with D


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Figure 1. Transmission electron microscopy images of (A,B) Stöber SNPs with an average diameter of 357 ± 22.8 nm (SNE), (C,D) etched Stöber SNPs with an average diameter of 357 ± 22.8 nm (SE), and (E,F) rattle shape SNPs with an average shell diameter of 328 ± 11 nm, average core diameter of 163 ± 9.74 nm, and average shell thickness of 36.07 ± 3.96 nm (RL). Scanning electron microscopy images of (G,H) SNE, (I,J) SE, and (K,L) RL.

Figure 2. Nitrogen adsorption−desorption isotherms of (A) SNE, (B) SE, and (C) RL SNPs and table of determined surface area and cumulative volume of pores for each particle type. (Insets) Pore size distribution plots. Measurement of cumulative pore volume by nitrogen adsorption confirms the morphology of SNE, SE, and RL. The pore volume of RL particles was significantly higher than that of SNE and SE, confirming the presence of an interior empty space. Likewise, the SE pore volume was higher than SNE pore volume, confirming the etched surface on SE particles.

FV1000 confocal microscope (Olympus Corporation, Shinjuku, Tokyo, Japan) at 60× magnification. For colocalization studies, cells were seeded on two chambered cover glasses (Lab-Tek Chambered #1.0 Borosilicate Coverglass System), allowed to grow for 24 h in an incubator, and washed with PBS. Cells were then treated with FITC-doped nanoparticles for 15 min and 4 h

in two different conditions of static and dynamic conditions and washed twice with PBS. LysoTracker Deep Red (50 nM) with RPMI media was then added to each chamber and incubated for 1 h. Next, Hoechst (2 μg mL−1) was added and incubated for an additional 10 min. The remaining staining solutions were removed by three rounds of cell washing with PBS. The E


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Table 1. Hydrodynamic Diameter, Zeta Potential, TEM Diameter, and Dry and Wet State Density Values of Synthesized SNPs hydrodynamic diameter (nm)

Stöber Stöber etched Stöber etched + rattle

zeta potential (mV)

size by TEM

dry state density (g/mL)

wet state density (g/mL)

water

RPMI

RPMI and 10% serum

water

RPMI

RPMI and 10% serum

378.3 ± 5.4 431.6 ± 6.4

402.6 ± 7.3 462.3 ± 9.6

566.6 ± 17.6 535.1 = 16.7

−32.1 ± 2.6 −22.6 ± 1.5

−27.8 ± 1.2 −24.1 ± 1.7

−10.5 ± 1.7 −8.7 ± 0.7

357 ± 22.8 357 ± 22.8

1.90 ± 0.1 1.83 ± 0.1

1.95 ± 0.1 1.91 = 0.1

443 ± 6.2

469 ± 5.8

588 ± 18.2

26.8 ± 0.3

14.5 ± 0.6

−9.7 ± 0.9

357 ± 22.8

1.67 ± 0.3

1.83 ± 0.1

411.0 ± 1.9

522.0 ± 1.7

548.6 ± 13.9

28.3 ± 0.5

16.7 ± 0.7

−10.1 ± 0.8

328 = 11.0

1.09 ± 0.2

1.54 = 0.1

Figure 3. Cell density, differential proliferation, and cell morphology under static and dynamic conditions. (A) Cell density at 0, 1, 2, and 3 days of incubation was measured in the presence and absence of dynamic flow. (B) A Click-iT EdU assay was used to incorporate EdU to DNA during synthesis by click chemistry to determine the rate of cell proliferation by means of fluorescent detection under both dynamic and static conditions. (C) Morphology of RAW264.7 macrophages treated with subtoxic concentrations of SNPs exposed to static and dynamic conditions was studied by applying F-actin stain and imaging by confocal microscopy.

nanoparticles allows for loading of bioactive agents in the hollow interior surrounding the core as well as drug release via mesopores in the shell. Nanorattles provide significant advantages over other nanostructures due to their high surface area and increased loading capacity. Comparing TEM and SEM images of SNE (Figure 1A,B,G,H) and SE particles (Figure 1C,D,I,J) the increased roughness of SE SNPs is not clearly obvious. However, increased surface roughness is confirmed by increased cumulative volume of pores and surface area utilizing nitrogen adsorption−desorption measurements shown in Figure 2. The adsorption isotherms according to IUPAC classification for SNE and SE were type II, confirming a solid nonporous structure, while RL SNPs showed typical type IV isotherms, revealing a porous structure.36 RL SNPs had a 42 m2g−1 surface area, almost 4-fold more than the 10.73 m2g−1 surface area of SE SNPs due to the porous shell and hollow structure. The hydrodynamic diameters measured by DLS, zeta potential, diameters measured by TEM, and dry and wet

excitation wavelengths for FITC, Hoechst, and LysoTracker Deep Red were adjusted to 495, 350, and 647 nm, respectively. The intensities of the photodetector sensitivity and the laser beam were kept constant to enable comparison of the relative fluorescence intensities between experiments. Statistical Analysis. At least three repeats for each data point were performed. Results are expressed as mean ± SD. Statistical significance was assessed by one-way ANOVA, and a Tukey post-test correction was performed where a difference was detected. The results are considered significant compared to control at p < 0.05.

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RESULTS Synthesis and Characterization of SNPs. The morphology and size of each particle were evaluated by SEM and TEM (Figure 1). The rattle-like hollow structure of RL SNPs was confirmed, with an average shell diameter of 328 ± 11 nm, average core diameter of 163 ± 9.74 nm, and average shell thickness of 36.07 ± 3.96 nm. The structure of rattle F


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Table 2. Diffusion and Sedimentation Coefficients, Volume Fraction, Apparent Density, Drag Coefficient, and Apparent Mass of Hydrated SNPs Used To Calculate Sedimentation and Diffusion Velocities silica nanoparticles

D (×10−12m2/s)

S (×10−9 s)

Vf

ρs,a (kg/m3)

f (×10−9kg/s)

ma (×10−16kg)

Stöber Stöber etched Stöber etched + rattle

1.00 1.06 0.97 1.04

5.30 5.37 4.46 2.41

0.25 0.30 0.22 0.21

1238.01 1270.23 1185.76 1115.41

4.27 4.03 4.43 4.14

1.18 1.02 1.26 0.96

state density values of synthesized SNPs are summarized in Table 1. The hydrodynamic diameter and zeta potentials of SNPs in DI water, RPMI media, and RPMI with 10% FBS were determined by DLS and Zeta Sizer to reveal the influence of the protein corona and ionic strength on particle size and charge. Assessing the Effective Density of Synthesized SNPs. To accurately characterize the dosimetry of ENMs in in vitro toxicological experiments, one should consider the determining factors such as the sedimentation and diffusion velocities of particles in cell culture media. The effective density of ENMs directly influences the particle’s sedimentation on the surface of cells. The effective density of nanoparticles can be obtained utilizing a combination of analytical ultracentrifugation and dynamic light scattering techniques. DeLoid et al. recently introduced a simple and fast procedure of volumetric centrifugation (VCM) using packed cell volume (PCV) tubes and benchtop centrifugation to measure the effective density of particles.35 By applying this method, we have measured the effective density of synthesized SNPs in both the dry and wet state. Pellet volumes were measured, and using the total mass of silica in each PCV tube, the density was calculated. It was assumed that, during centrifugation, pellets formed in a manner corresponding to random close stacking. Under aqueous conditions, media is adsorbed inside the RL particles as well as onto the surface of all particle types. The adsorption of media and proteins on the particles and specifically inside the hollow section of rattle structured SNPs can effectively change the densities of these particles due to the mass added by media adsorption compared to the dry state. In order to account for protein and media adsorption and the corresponding change in density, pellet mass and volume (MENM and Vpellet, respectively) were obtained via particle centrifugation in PCV tubes. The stacking factor (SF) of the pellet formed during centrifugation was presumed to be 0.634, corresponding to random close stacking. As shown in Table 1, the measured densities were 1.95, 1.91, 1.83, and 1.54 g/mL for SNE, SE, SE+, and RL SNPs, respectively. Between SNE, SE, and SE+, no significant difference in the densities of solid nonhollow SNE, SE, and SE+ were observed; however, RL particles had a significantly lower density than the other three particle types. Comparing the measured wet and dry densities for each particle type, we observed slight differences for solid particles; however, RL particles showed a more noticeable change. For solid particles, increased density in the wet state occurs due to adsorption of media and formation of the protein corona only on the surface of particles. However, this phenomenon is more pronounced for hollow structured rattle shape particles due to infiltration of media into the hollow section of particles through porous shell. Influence of Dynamic and Static Conditions on Cell Proliferation and Morphology. We simulated dynamic conditions by placing cell culture plates on a tilting platform. Cell density, differential proliferation, and cell morphology under static and dynamic conditions were examined to study the influence of dynamic flow on cells before treatment with

SNPs (Figure 3). Cell viability up to 3 days for both conditions was not significantly different when the cells were cultured in the absence of particles. To validate cell density results, the rate of cell proliferation was determined by the DNA synthesis rate using the Click-iT EdU assay under both dynamic and static conditions. The main goal of investigating the morphology of the cells treated with subtoxic concentrations of SNPs exposed to static and dynamic conditions was to evaluate the capability of the dynamic flow in rearranging the cells by making them more aligned under shear forces. As shown in Figure 3C, dynamic flow had no significant effect on changing the morphology of cultured cells. Dynamic conditions compared to static conditions did not impact cell proliferation or morphology. This rules out the effect of flow on cells in subsequent studies. Sedimentation (Vs) and Diffusion (VD) Velocities of Synthesized SNPs. The diffusion coefficients (D) of SNPs were calculated using the Stokes−Einstein equation D = KBT /3πηdh

(2)

where values for the Boltzmann constant (KB), absolute temperature (T), and media viscosity (η) were 1.38065e−23 J/K, 310.15 K, and 8.0e−4 kg/m·s, respectively. Hydrodynamic diameters (dh) of particles in media were determined using DLS. Diffusion velocities (VD) were calculated using eq 3, derived from the Einstein equation, in which x is the distance that particles traveled via diffusion. The value for x was considered in our calculations to be 2.21e−3 mm, that is, the average distance particles should travel to be exposed to cells during cell culture experiments. VD = 2D/x

(3)

Sedimentation velocity (Vs) was obtained using eq 4. Values for media density (ρm), acceleration due to gravity (g), and media viscosity (η) were 1000 kg/m3, 9.81 m/s2, and 8.0e−4 kg/m.s, respectively. Particle densities (ρs) in the wet state were considered in these calculations for each particle type.29,35 ⎛ d ⎞2 Vs = 2g (ρs − ρm )⎜ h ⎟ /9η ⎝2⎠

(4)

For particle density (ρs) measurements, protein corona formation due to nonspecific adsorption should also be considered. To address this issue, the apparent density of particles (ρs,a) in cell culture media was calculated by evaluating the volume fraction (Vf) of each SNP type based on eq 5. Vf was calculated based on having the actual volume of particles (diameter obtained by TEM) and hydrodynamic volume (using dh). ρs,a = 1 × (1 − Vf ) + ρs × Vf

(5)

Sedimentation coefficients of SNPs were calculated using the Manson−Weaver equation, where m is the mass of SNPs, f is G


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Figure 4. (A) Sedimentation (Vs) and diffusion (VD) velocities for each type of prepared SNP. (B) Ratio of sedimentation and diffusion velocities (Vs/VD) to assess the dominant factor in particle transport toward cell surface.

Figure 5. RAW264.7 macrophages treated with SNPs and incubated for 24 h to evaluate corresponding cytotoxicity. (A) Cell viability of treated RAW264.7 macrophages using a CCK-8 assay as a function of mass concentration. Cytotoxicity studies under static and dynamic conditions demonstrated increased cell viability under dynamic conditions. (B) LC50 values obtained for each type of SNP under static and dynamic conditions are presented in the table. (C) Integrity of RAW264.7 macrophage plasma membrane after 24 h of incubation with different concentrations of SNPs studied by a lactate dehydrogenase (LDH) release assay under static conditions. (D) LDH studies under dynamic conditions with different concentrations of SNPs.

drag coefficient, ρs is the particle density, and ρm is cell culture media density. Drag coefficients ( f) were obtained by eq 7. ⎛ ρ ⎞ S = m /f × ⎜⎜1 − m ⎟⎟ ρs ⎠ ⎝

f=

kBT D

ma = ρs,a × hydrodynamic volume of SNPs

(8)

Table 2 summarizes all different parameters calculated to obtain sedimentation (Vs) and diffusion (VD) velocities. With the use of eqs 2−7, VD and Vs values were calculated to clearly exhibit the difference in the sedimentation rate of particles with different densities and consequent cell uptake. As shown in Figure 4, there is no significant difference in VD of all four types of SNE, SE, SE+, and RL particles (726, 768, 700, and 749 × 10−12 m/s respectively). However, values increased in the following order: RL SNPs < SE+ SNPs < SNE SNPs < SE SNPs. In Vitro Cytotoxicity. The cellular toxicity of the nanoparticles was assessed under both static and dynamic conditions in RAW 264.7 macrophages in a range of

(6)

(7)

To consider the protein corona and media around each particle in calculations, m was converted to apparent ma, resulting in increased values compared to the dry state. Apparent ma was determined using the apparent density of particles (ρs,a) in eq 8.29 H


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Molecular Pharmaceutics concentrations (8−250 μg mL−1). RAW 264.7 cells were chosen due to their high phagocytic activity, resembling the macrophage response to ENM exposure. Concentrationdependent, density-dependent, and cell culture conditiondependent toxicity effects of particles were observed using CCK-8 and LDH assays (Figure 5). In Vitro Cell Uptake and Colocalization. Cell uptake and intracellular colocalization were evaluated using TEM, ICP-MS, flow cytometry, and confocal laser scanning microscopy (Figures 6 and 7). Subtoxic concentrations were selected in

Figure 6. TEM images of untreated RAW264.7 macrophages (control) or those treated with SNPs for 4 h under static and dynamic conditions. Cell uptake of SNPs and localization inside vesicles were observed. A higher number of particles were observed under static conditions as compared to dynamic conditions. Red arrows point to particles inside cells. Particles were not observed inside the nucleus.

Figure 7. (A) Confocal microscopy images of RAW 264.7 macrophages treated with FITC-doped nanoparticles under static and dynamic conditions for 15 min and 4 h. Lysosomes are shown in red utilizing LysoTracker Deep Red, and particles are shown in green. (B) ICP-MS and (C) flow cytometry analyses of treated RAW 264.7 macrophages with solid nonhollow and hollow rattle structure SNPs under the exact same conditions as those in the cytotoxicity studies to evaluate particle cell associations based on number of particles per cell. Data are expressed as mean ± SD from (n = 3), and **P < 0.05.

all experiments to eliminate cell death. Nontreated cells were used as controls. Figure 6 shows that macrophages were taken up by nanoparticles in both static and dynamic conditions. As seen in Figure 6C, particles localized in the endosomal or endolysosomal vacuoles. Time-dependent cell uptake was confirmed in both cell culture conditions using confocal microscopy (Figure 7A). A relatively lower number of particles was observed under dynamic conditions versus static conditions as indicated by both TEM and confocal microscopy images. However, to quantify these data, we conducted flow cytometry and ICP-MS (Figure 7B,C).

Initially, density’s effect on cytotoxicity was to be evaluated using silica nanorattles and typical solid Stöber particles. Density, however, was not the only characteristic that differed between these particles. Surface charge and roughness were altered during the etching process for RL particles, resulting in a rough and positively charged outer shell, compared to the nonporous negatively charged surface of Stö ber (SNE) particles. Thus, two more types of silica nanoparticles were included: silica etched (SE), with an etched surface similar to RL and negative zeta potential similar to SNE, and silica etched amine-terminated (SE+), with an etched surface and positive charge similar to RL (Scheme 1). The inclusion of these additional particle morphologies allowed for the effects of surface charge and surface porosity to essentially be eliminated during the study of the density effect. Nonspecific protein adsorption on particles was confirmed by increased hydrodynamic diameter on all SNPs. Zeta potential measurements revealed highly negatively charged SNE and SE particles, rendering colloidal stability in aqueous medium, while RL particles showed positive charge due to the amine groups in middle layer outside of the mesoporous shell (Table 1).34 A successful amine-termination reaction to convert negatively charged SE to positively charged SE+ particles was detected by a change in zeta potential from −22 to +26.8 in water. The higher ionic strength of RPMI media compared to water resulted in a screening effect and decreased the Debye length, which in turn decreased positive charges and increased

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DISCUSSION Solid nonporous spherical Stöber SNPs (SNE) with average diameter of 357 ± 22.8 nm were previously synthesized and stored in ethanol (Figure 1).11,33 The synthesis of silica nanorattles was adapted from Chen et al.34 The process relies on a layer-by-layer approach and selective etching, in which FITC-doped cores are first synthesized, followed by an organic/inorganic silica hybrid layer, followed by a solid silica shell (Scheme 2). Each layer essentially followed a modified Stöber method, in which TEOS and/or TSD was condensed in the presence of water and ammonia. This resulted in a middle layer with a silica framework of lesser density. The less-dense middle layer was etched out using concentrated hydrofluoric acid, leaving the FITC-doped solid core inside a hollow interior, surrounded by a porous shell. I


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in which low density RL particles showed lower cell uptake compared to higher density SE+ particles (Figures 6 and 7). The theoretical velocity of sedimentation calculations indicates a higher probability of gravitational settling for higher density particles. The cytotoxicity and cellular uptake of silica nanoparticles of various densities on RAW 264.7 macrophages was evaluated under both static and dynamic cell culture conditions to assess any potential impact of density and flow conditions on toxicity profiles of ENMs. Experimental conventional static well plate cell culture nanotoxicity studies showed lower toxicity for less-dense silica nanorattles compared to more-dense Stöber particles with the same size, surface roughness, and charge. Particles exposed to flow on a tilting shaker for both high and low density constructs show less sedimentation and more-homogeneous distribution with little to no toxicity up to 250 μg/mL. An important issue for in vitro ENM toxicity assessment is determining the effective dosimetry. Generally, dosing is reported by particle concentration, assuming a homogeneous distribution of nanoparticles in cell culture media. However, important parameters such as particle effective density, sedimentation and diffusion coefficients, and velocity of sedimentation are often ignored in nanotoxicity studies. Wittmaack et al. highlighted the influence of gravitational settling of nanoparticles and physical overload effects on cells, suggesting misinterpreted cytotoxicity assessments by ignoring the sedimentation effect. Under static conditions, nanoparticles tend to sediment onto cell surfaces due to gravitational force, which in turn could induce physicochemical stress in the cell and increase effective dosimetry of nanoparticles exposed to cells.37,38 Herein, decreased sedimentation, cell uptake, and toxicity for lower density particles and/or presence of flow were validated through theoretical and experimental evaluations. For future in vitro nanoparticle cytotoxicity studies, these and other results in the literature imply the need to consider the sedimentation of ENMs, in which particle suspensions would be under flow conditions and with different particle densities. Introducing flow to an in vitro cellular assay utilizing dynamic conditions provides a homogeneous distribution of ENMs in culture media, ensures consistent cytotoxicity results from different laboratories, and brings in vitro nanotoxicity evaluations one step closer to correlation with in vivo studies.

negative charges. Protein corona formation is also detectable by zeta charge change toward the neutral range (−10 to 10 mV) upon interaction with proteins in the media containing FBS. Figure 3 shows a comparison of the cells under dynamic conditions to those under static conditions where there was no significant difference in the overall cell proliferation and morphology. Thus, cell culture conditions did not impact cell viability during cytotoxicity or cellular uptake studies. The morphology of RAW264.7 cells after exposure to static conditions, dynamic conditions, and subtoxic concentrations of SNPs was evaluated using F-actin stain and imaged by confocal microscopy. RAW264.7 cells in all conditions showed a randomly aligned morphology, and no alignment under dynamic conditions to the direction of flow was observed, emphasizing the absence of organization of the cell cytoskeleton under shear stress in dynamic conditions. It is noteworthy that the Vs value of RL SNPs with lowest density due to the hollow structure is significantly lower than other types of solid particles, as shown in Figure 4. The ratio of sedimentation to diffusion velocities is directly in correlation with the cellular uptake of nanoparticles due to the change in particle concentration within the interaction zone of cells, altering the number of particles exposed to the cell surface. Solid nonhollow SNPs with a higher Vs/VD could sediment quickly and enter the interaction zone with cells, resulting in an increased dosimetry of particles exposed to cells compared to a slower sedimentation of low density hollow structure RL particles with low sedimentation to diffusion velocities ratio at the same dosing concentrations. Mass concentration dosimetry was used in toxicity studies. Within the same mass concentrations, a higher total number of RL particles was delivered relative to the other particle types due to a lower density of RL SNPs. Under static cell culture conditions, cells exposed to RL particles demonstrated a lower degree of toxicity compared to SNE, SE, or SE+ SNPs (Figure 5). With use of the CCK-8 assay, the LC50 values for RL particles was 197 μg/mL, whereas for SNE, SE, and SE+, the LC50 values were 86, 92, and 107 μg/mL, respectively. These results correlate with the sedimentation to diffusion velocities ratio. This was further confirmed by cytotoxicity evaluation using LDH release. At each concentration, RL particles caused the least amount of LDH release relative to all other particles. It can thus be assumed that density plays a significant role in silica nanoparticle cytotoxicity as measured under typical static cell culture conditions due to density’s effect on particle sedimentation. The cells in the static condition probably experience an increased concentration of solid nonhollow SNE, SE, and SE+ SNPs than that of RL SNPs if the solid dense SNPs sedimented under the gravitational force. Conversely, under dynamic cell culture conditions, none of the different types of SNPs showed a toxic effect up to 250 μg mL−1. An insignificant difference in toxicity profiles among particle types suggests media agitation in dynamic conditions disrupted particle sedimentation; as such, particles interacted to a lesser extent with the cells. Disrupted sedimentation reduces the nanoparticle concentration in the interaction zone of cells, resulting in lower uptake and less toxicity. We evaluated the amount of SNPs associated with RAW264.7 macrophages using ICP-MS. The data was further validated by flow cytometry. The higher uptake of particles in the static condition compared to the dynamic condition with TEM and confocal microscopy was consistent with flow cytometry and ICP-MS results for FITC-doped nanoparticles,

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CONCLUSION In vitro assessment of ENM toxicity is by and large performed under static conditions assuming homogeneous distribution in cell culture media. Herein, effects of density and flow on toxicity and cellular uptake of SNPs in RAW 264.7 macrophages were studied with particles under conventional static cell culture conditions, and dynamic conditions facilitated by a tilting shaker. We observed slightly decreased sedimentation, cell uptake, and toxicity for lower density particles and under flow conditions. We conclude that ENMs’ physicochemical properties, such as density, along with flow conditions of cell culture environment influence sedimentation and effective dosimetry presented to the interaction zone of cells, which in turn change cell uptake and cytotoxicity.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (H.G.) J


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Hamidreza Ghandehari: 0000-0002-9333-9964 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this project was provided by the National Institute of Environmental Health Sciences of the NIH (R01ES024681), the University of Utah College of Pharmacy Skaggs Graduate Research Fellowship (M.Y.), and the Undergraduate Research Opportunities Program Assistantship (Z.B.B.). This work made use of the University of Utah shared facilities of the Micron Microscopy Suite and the University of Utah USTAR shared facilities supported in part by the MRSEC Program of the NSF under Award No. DMR-1121252.

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Influence of Silica Nanoparticle Density and Flow Conditions on

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