Influence of Silica Nanoparticle Density and Flow Conditions on

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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 Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00213 • Publication Date (Web): 02 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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Molecular Pharmaceutics

Influence of Silica Nanoparticle Density and Flow Conditions on Sedimentation, Cell uptake and Cytotoxicity Mostafa Yazdimamaghani 1, 2, Zachary B. Barber 3, Seyyed Pouya Hadipour Moghaddam1, 2, Hamidreza Ghandehari 1, 2,3,* 1

Utah Center for Nanomedicine, Nano Institute of Utah, 2Department of Pharmaceutics and

Pharmaceutical Chemistry, 3Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA * Hamidreza Ghandehari, Email: [email protected],

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Graphical abstract

Highlights - Theoretical velocity of sedimentation calculations indicate higher probability of gravitational settling for higher density particles - Experimental conventional static well-plate cell culture nanotoxicity studies showed lower toxicity for less-dense silica nanorattles compared to denser Stöber particles with similar size, surface roughness and charge - Both high and low density particles when exposed to flow on tilting shaker showed less sedimentation and more homogeneous distribution with little to no toxicity up to 250 µg/mL concentration


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Abstract Careful evaluation of 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 ENMs 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 350nm 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 non-homogeneous 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. 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 volume of media. Under static conditions nanoparticles tend to sediment due to gravitational force 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 diameter and density of particles as well as flow rate, and viscosity.16-20 It has been reported that for particles larger than 100nm, 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. 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.


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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 ENMs delivered dose 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 influence of ENM density and sedimentation effect on cell uptake and toxicity.15, 29-30 Using microfluidic devices to generate a concentration gradient of nanoparticles on cultured cells, toxicity of quantum dots (QD) was evaluated.15 Avoiding gravitational settlement of QDs by flow in microfluidics chips provided a homogeneous distribution of nanoparticles and led to increased percent of viable cells compared to static conditions.15 One challenge with the use of microfluidic systems with 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 use of inverted cell culture conditions. Cho et al. reported the uptake of gold nanoparticles with different sizes, shapes, and density on 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 effective concentration of particles. Further cells can migrate from inverted section to the bottom of wellplate, 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 sedimentation effect on in vitro toxicity. This method is simple, inexpensive, and reproducible. To further optimize the system one could add shear measurement devices to elaborate the influence of tilting speed on sedimentation effect. In addition, there is 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 Stöber SNPs with approximately 350nm in diameter were synthesized and characterized. Surface roughness and charge were modified to obtain the same surface properties as low density hollow structured SNPs. To evaluate the effect of density and flow on toxicity and cellular uptake of SNPs,


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Raw264.7 macrophages were treated with particles under conventional static cell culture condition, and dynamic condition facilitated by a tilting shaker.

Scheme 1. (A) Spherical particles of similar size were prepared with different surface roughness, charge, and density while controlling for other differences between particle types to examine 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 Stober 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.



Materials and Methods Materials. N-[3-(trimethoxysilyl) propyl]ethylenediamine (TSD), tetraethyl orthosilicate (TEOS, 98%), Triton™ X-100, (3-Aminopropyl)triethoxysilane (APTES), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Ammonium hydroxide (NH4OH, 28–30% as NH3) and hydrofluoric acid (HF 48%) were obtained from EMD Millipore Corporation (Billerica, MA, USA). Absolute ethanol (200 proof) was purchased from Decon Laboratories, Inc. (King of Prussia, PA, USA). Trypan blue stain 0.4% was received from Invitrogen (Carlsbad, CA, USA). RAW 264.7 macrophages (ATCC® TIB-71™) were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA). 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, USA). Cell counting kit-8 (CCK-8) cytotoxicity assay was received from Dojindo (Rockville, MD, USA). ActinRed 555 ReadyProbes reagent, Hoechst, lactate dehydrogenase (LDH) assay, LysoTracker Deep Red, and fluorescein isothiocyanate (FITC) were received from Thermo Fisher Scientific (Eugene, OR, USA). All chemicals were used as received without further purification.

Synthesis of silica nanoparticles Nonporous SNPs with 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 absolute ethanol, and 2 mL Tetraethyl orthosilicate (TEOS). Solution B contained 60 mL


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absolute ethanol and 20 mL Ammonium Hydroxide (28% aq). Solution C contained 8 mL absolute ethanol and 200 µL N-[3-(trimethoxysilyl) propyl]ethylenediamine (TSD). Solution B was placed in an Erlenmeyer flask and stirred at 400 rpm at room temperature. 5 mL solution A 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 solutions were allowed to stir for 3 hrs. Particles were then washed with DI H2O three times. In the second step prepared particles were etched using HF. One mL of particles suspended in DI H2O at a concentration of 9 mg/mL was placed in a 1.5 mL microcentrifuge tube. 15 µL of 10% HF (aq) was added to the particles. At a setting 7/10 of maximum vortex rate, particles were vortexed for 5 minutes to facilitate etching. Scheme 2 further illustrates the steps for the synthesis of RL SNPs.

Scheme 2. 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.



Surface-etched Stöber particles (SE) were synthesized by etching pre-fabricated Stöber particles (SNE) under precisely the same concentration and conditions as the RL SNPs. Amine terminated surface-etched Stöber particles (SE+) were synthesized using a portion of the SE particles. 20 mL SE particles suspended in absolute ethanol at a concentration of approximately 10 mg/mL were added to a round bottom flask, along with 80 mL absolute ethanol. Solution was stirred at 500 rpm under anhydrous conditions, obtained using an N2-filled balloon. 4.2×10-3M of (3Aminopropyl)triethoxysilane (APTES) was added. Solution was stirred 24 hrs, then washed using absolute ethanol. 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 supernatant 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 scanning electron microscopy (SEM) with a FEI Quanta 650 FE-SEM (Hillsboro, OR, USA) operating at 20 kV were used to characterize morphology and size of prepared nanoparticles. Malvern Instruments Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, U.K.) was utilized to measure hydrodynamic diameter and zeta potential of synthesized SNPs. 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, USA) for determining surface area and pore size. Theoretical sedimentation velocities were calculated from particle densities using sedimentation velocity equation.


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Particle density measurement Density of silica nanoparticles was measured using centrifugation in packed cell volume (PCV) tubes.35 Pellet volumes were measured using PCV, and knowing total mass of silica in each tube 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. 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. Corresponding change in density in wet state was calculated using equation 1 where media density (ρmedia) of 1 g/mL, and silica density (ρENM) of 2 g/mL were used and pellet mass and volume (MENM and Vpellet respectively) were obtained via particle centrifugation in PCV tubes. = + [

1 −

!]

(1)

Cell Culture and Cytotoxicity Assay Cell density was investigated by counting cell number utilizing Countess® Automated Cell Counter (Thermo Fisher Scientific Corporation, Grand Island, NY, USA) 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 three 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 six counts. Cell proliferation was investigated by the Click -iT EdU microplate assay kit (Thermo Fisher Scientific) following manufacturer provided protocols. DNA synthesis measured by 11 ACS Paragon Plus Environment


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 excitation/emission wavelength of 490/585 nm utilizing SpectraMax® M2 (Molecular Devices, Sunnyvale, CA, USA) 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 washed with PBS twice. RPMI media with 10% fetal bovine serum providing varying concentrations of 8 µg/mL to 250 µg/mL of SNPs were incubated with cells up to 24 h. For control samples fresh media was added without SNPs. Then, media was aspirated and the cells were washed twice with PBS. Cell viability was determined with CCK-8 assay and membrane integrity was evaluated by 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 Spectroscopy (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


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sheets and incubated for 24h at 37 °C in 5% CO2. Next, cells were washed and treated with LC50 concentration of different types of SNPs and incubated for 4 h under two different conditions of static and laminar flow. Then, cells were washed 3 times with PBS and fixed with 1 mL of ﬁxing solution (2.5% glutaraldehyde + 1.0% paraformaldehyde). Cells were post-fixed 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 JOEL 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 spectroscopy (Santa Clara, CA, USA). For ICP-MS measurements cells were treated with SE+ and RL SNPs with the same concentration of 100 µg/ml and incubated for 24 hours 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. Cell number for each time point was determined for each particle type. Next, cells were washed 3 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 oC on the hot plate for 2 hr. The oxidized product was dissolved in 2 mL water and transferred into 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 ICPMS instrument was used under operating conditions suitable for routine multi-element analysis. All the chemicals used were of trace metal grade. The number of SNP association 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, 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 co-localization To test the influence of the dynamic condition on cell morphology changes, in the absence of SNPs, cells were seeded, incubated, and washed on a 6 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 Olympus FluoView 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 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 additional 10 min. The remaining staining solutions were removed by three times of cell washing with PBS. The 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.


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Statistical Analysis At least three repeats for each data point were performed. Results are expressed as mean ± S.D. Statistical significance was assessed by one-way ANOVA, and Tukey post-test correction was performed where a difference was detected. The results are considered significant compared to control at p < 0.05.

Results Synthesis and characterization of SNPs Morphology and size of each particle were evaluated by SEM and TEM (Figure 1). Rattle-like hollow structure of RL SNPs was confirmed, with 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 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.



Figure 1. Transmission electron microscopy images of (A-B) Stöber SNPs with average diameter of 357±22.8 nm (SNE), (C-D) Etched stöber SNPs with average diameter of 357±22.8 nm (SE), and (E-F) Rattle shape SNPs with 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.

Comparing TEM and SEM images of SNE (Figure 1A, B, G, and H) and SE particles (Figure 1C, D, I, and 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 solid nonporous structure, while RL SNPs showed typical type IV isotherms, revealing porous


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structure.36 RL SNPs had a 42 m2g-1 surface area, almost 4-fold more than 10.73 m2g-1 surface area of SE SNPs due to porous shell and hollow structure.

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, SE pore volume was higher than SNE pore volume, confirming the etched surface on SE particles.

The hydrodynamic diameters measured by DLS, zeta potential, diameters measured by TEM, and dry and wet 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 influence of protein corona and ionic strength on particle size and charge.

Assessing the effective density of synthesized SNPs To accurately characterize dosimetry of ENMs in in vitro toxicological experiments, one should consider the determining factors such as sedimentation and diffusion velocities of particles in cell culture media. Effective density of ENMs directly influence the particle’s sedimentation on the surface of cells. 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 method (VCM) using packed cell volume (PCV) tubes and benchtop centrifugation to measure effective density of particles.35 By applying this method we have measured effective density of synthesized SNPs in both dry and wet state.

Pellet volumes were measured, and using the total mass of silica in each PCV

tube, 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. 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 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, 18 ACS Paragon Plus Environment

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SE+, and RL SNPs, respectively. Between SNE, SE, and SE+, no significant difference in densities of solid non-hollow 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 wet state occurs due to adsorption of media and formation of 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.

Table 1. Hydrodynamic diameter, zeta potential, TEM diameter, and dry and wet state density values of synthesized SNPs

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, rate of cell proliferation was 19 ACS Paragon Plus Environment


determined by DNA synthesis rate using Click-iT EdU assay under both dynamic and static conditions. The main goal of investigating the morphology of the cells treated with sub-toxic concentrations of SNPs exposed to static and dynamic conditions was to evaluate the capability of the dynamic flow in re-arranging 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 condition compared to static condition did not impact cell proliferation or morphology. This rules out the effect of flow on cells in subsequent studies.

Figure 3. Cell density, differential proliferation, and cell morphology under static and dynamic conditions. (A) Cell density at 0, 1, 2, and 3 days 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 rate of cell


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proliferation by means of fluorescent detection under both dynamic and static conditions. (C) Morphology of RAW264.7 macrophages treated with sub-toxic concentrations of SNPs exposed to static and dynamic conditions was studied by applying F-actin stain and imaging by confocal microscopy.

Sedimentation (Vs) and diffusion (VD) velocities of synthesized SNPs Diffusion coefficients (D) of SNPs were calculated using the Stokes-Einstein equation: # = $% &/3)*+,

(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 equation 3 derived from Einstein equation in which x is the distance which particles travelled via diffusion. The value for x was considered in our calculations 2.21e-3mm that is the average distance particles should travel to be exposed to cells during cell culture experiments. -. = 2#/0

(3)

Sedimentation velocity (Vs) was obtained using equation 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 wet state were considered in these calculations for each particle type.29, 35

-1 = 2231 − 43 5 46 /9* 6

(4)

For particle density (ρs) measurements protein corona formation due to non-specific adsorption should also be considered. To address this issue apparent density of particles (ρs,a) in cell culture media was calculated by evaluating the volume fraction (Vf) of each SNP type based on equation 5. Vf was calculated based on having the actual volume of particles (diameter obtained by TEM) and hydrodynamic volume (using dh). 21 ACS Paragon Plus Environment


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1, = 1 × :1 − -; < + 1 × -;

(5)

Sedimentation coefficients of SNPs were calculated using Manson-Weaver equation where m is the mass of SNPs, f is drag coefficient, ρs is the particle density, and ρm is cell culture media density. Drag coefficients (f) were obtained by equation 7. = = >/? × 31 − ?=

@ @A

4

BC D .

(6) (7)

To consider protein corona and media around each particle in calculations, m was converted to apparent ma resulting in increased values compared to dry state. Apparent ma was determined using apparent density of particles (ρs,a) in equation 8.29 > = ρ1. × GH+IJ+HKL>MN OJPQ>R J? =STU

(8)

Table 2 summarizes all different parameters calculated to obtain sedimentation (Vs) and diffusion (VD) velocities. Using equations 2 to 7, VD and Vs values were calculated to clearly exhibit the difference in 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 ×10-12m/s respectively). However, values increased in the following order: RL SNPs < SE+ SNPs < SNE SNPs < SE SNPs.

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.


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

In vitro cytotoxicity Cellular toxicity of the nanoparticles was assessed under both static and dynamic conditions in RAW 264.7 macrophages in a range of concentrations (8–250 µg mL–1). RAW 264.7 cells were chosen due to their high phagocytic activity, resembling macrophage response to ENM exposure. Concentration-dependent, density- dependent, and cell culture condition-dependent toxicity effects of particles were observed using CCK-8 and LDH assays (Figure 5).



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 CCK-8 assay as a function of mass concentration. Cytotoxicity studies under static and dynamic conditions demonstrated increased cell viability under dynamic condition. (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 incubation with different concentrations of SNPs studied by lactate dehydrogenase (LDH) release assay under static condition. (D) LDH studies under dynamic condition with different concentrations of SNPs.

In vitro cell uptake and co-localization Cell uptake and intracellular co-localization were evaluated using TEM, ICP-MS, flow cytometry, and confocal laser scanning microscopy (Figures 6 and 7). Sub-toxic concentrations were selected in all experiments to eliminate cell death. Non-treated cells were used as controls. 24 ACS Paragon Plus Environment

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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). Relatively lower number of particles were observed under dynamic condition versus static condition as indicated by both TEM and confocal microscopy images. However, to quantify these data we conducted flow cytometry and ICP-MS (Figure 7B, C).



Figure 6. TEM images of untreated RAW264.7 macrophages (control), or 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 condition compared to dynamic condition. Red arrows point particles inside cells. Particles were not observed inside the nucleus.


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Figure 7. (A) Confocal microscopy images of RAW 264.7 macrophages treated with FITC-doped nanoparticles under static and dynamic conditions for 15 minutes and 4 hours. 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 non-hollow and hollow rattle structure SNPs under exact same conditions as cytotoxicity



studies were conducted to evaluate particle cell associations based on number of particles per cell. Data are expressed as mean ± S.D. from (n = 3), and ** P

Influence of Silica Nanoparticle Density and Flow Conditions on

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