Mechanosensitive Endocytosis of High-Stiffness, Submicron Microgels

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Mechanosensitive endocytosis of high-stiffness, sub-micron microgels in macrophage and hepatocarcinoma cell lines. Terra Kruger, Brittany E. Givens, Thiranjeewa Lansakara, Kendra Bell, Himansu Mohapatra, Aliasger K. Salem, Alexei V Tivanski, and Lewis L Stevens ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00111 • Publication Date (Web): 10 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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ACS Applied Bio Materials

Mechanosensitive endocytosis of high-stiffness, sub-micron microgels in macrophage and hepatocarcinoma cell lines. Terra M. Kruger1, Brittany E. Givens1,2, Thiranjeewa I. Lansakara3, Kendra J. Bell1, Himansu Mohapatra1, Aliasger K. Salem1, Alexei V. Tivanski3 and Lewis L. Stevens1,* 1. Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, The University of Iowa, Iowa City, IA 52242 2. Department of Chemical and Biochemical Engineering, College of Engineering, The University of Iowa, Iowa City, IA 52242 3. Department of Chemistry, The University of Iowa, Iowa City, IA 52242 ABSTRACT The mechanical properties of sub-micron particles offer a unique design space for advanced drug-delivery particle engineering. However, the recognition of this potential is limited by a poor consensus about both the specificity and sensitivity of mechanosensitive endocytosis over a broad particle stiffness range. In this report, our model series of polystyrene-co-poly(Nisopropylacrylamide) (pS-co-NIPAM) microgels have been prepared with a nominally constant monomer composition (50 mol% styrene and 50 mol% NIPAM) with varied bis-acrylamide crosslinking densities to introduce a tuned spectrum of particle mechanics without significant variation in particle size and surface charge. While previous mechanosensitive studies use particles with moduli ranging from 15 kPa to 20 MPa, the pS-co-NIPAM particles have Young’s moduli (E) ranging from 300-700 MPa, which is drastically stiffer than these previous studies as well as pure pNIPAM. Despite this elevated stiffness, particle uptake in RAW264.7 murine macrophages displays a clear stiffness dependence, with a significant increase in particle uptake for our softest microgels after a four hour incubation. Preferential uptake of the softest microgel, pS-co-NIPAM-1 (E = 310 kPa), was similarly observed with non-phagocytic HepG2 hepatoma cells; however, the uptake kinetics were distinct relative to that observed for RAW264.7 cells. Pharmacological inhibitors, used to probe for specific routes of particle internalization, identify actin- and microtubule-dependent pathways in RAW264.7 cells as sensitive to particle mechanics. For our pS-co-NIPAM particles at nominally 300 - 400 nm in size, this microtubule-dependent pathway was interpreted as a phagocytic route. For our highstiffness microgel series, this study provides evidence of cell-specific, mechanosensitive endocytosis in a distinctly new stiffness regime that will further broaden the functional landscape of mechanics as a design space for particle engineering.

Keywords: nanoindentation, microgel, mechanical properties, endocytosis, mechanosensitive, drug delivery * Corresponding author. Tel: (319)-335-8823; e-mail: [email protected]

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1. INTRODUCTION Successful delivery of a targeted drug-delivery vector requires overcoming multiple biological barriers, such as opsonization, splenic filtration and endocytosis, to enable prolonged circulation and controlled biodistribution.1-2 To address this issue, and improve their biological fate, physical and chemical modifications have been investigated to design particles with enhanced targeted delivery. These modifications including size, shape, charge, and the addition of targeting ligands on the particle surface each display an impact on cellular uptake in the drug delivery process3-4. Although much is known about these physical and chemical attributes of particles, relatively little is known about the effect of particle stiffness on cellular uptake5-6. Further investigation into this could lead to increased efficiency of tissue targeting and open a new route to the efficient delivery and control of cellular uptake. Previous studies have demonstrated that particle mechanics have significant influence over endocytic efficiency and modifying biodistribution in a wide range of particle materials and sizes, as well as in various cell types.7-11 One important demonstration of this mechanosensitive uptake response was done using soft and stiff polyacrylamide microbeads with two distinct bisacrylamide crosslinking concentrations7. As the particles were internalized through phagocytosis, there was no observable difference in uptake in bone marrow derived macrophages, despite a nearly three-fold difference in the Young’s modulus (E). However, when the particles were decorated with an IgG antibody, (to explore the potential of FcRmediated phagocytosis) there was a significant preference for the stiffer particles. This same trend was observed using poly(ethylene glycol) diacrylate (PEGDA) particles with a range of Young’s moduli from 0.255 kPa to 3.0 MPa12. Further, this uptake increase was even more pronounced in macrophage when compared to endothelial cancer cells. Finally, complementary in vivo studies have also shown that softer PEGDA particles frustrate phagocytosis in macrophages, increasing the circulation half-life of the particles12.These trends suggest particle softening may be leveraged as a useful tool to avoid macrophage clearance, increase circulation time, and provide for an increased therapeutic delivery9. Furthermore, a fundamental understanding of how cellular mechanosensitive endocytosis will contribute to its optimization of the particle physical properties that favor targeting and decrease rapid clearance. In contrast to these previous studies that display a consistent preference for stiffer particles, there has also been demonstration of the inverse effect. Other studies using hyaluronic acid templated on the surface of silica particles, showed that the softest, most deformable capsules (bending stiffness = 7.5 mN/m) were significantly more bound/internalized by HeLa cells, when compared to their stiffer counterparts with stiffness ranging from 17.6 – 28.9 mN/m 10. This pattern was also shown with poly(2-hydroxyethyl methacrylate) (pHEMA) particles (E = 15 – 156 kPa) and HepG2 hepatoma cells13. Again this inversely correlated pattern was observed, for alginate crosslinked nanolipogels under a wide range of moduli (E = 45kPa - 19MPa). As the stiffness of the nanolipogel increases, the uptake decreases in three different epithelial, adenocarcinoma cells. Further, when these nanolipogel were tested in vivo, there was no mechanosensitive variance in the biodistribution of these particles in any of the organs, except the tumor. This is highly significant for the potential to use mechanics as a tool for targeted drug delivery. With varying mechanosensitive patterns present in the literature, there is a distinct knowledge gap in understanding the mechanism behind this mechanosensitivity. The current literature contains studies with varying particle sizes, materials, and targeting ligands, as well as different cell types, making it difficult to draw distinct connections between these studies. It is likely that depending on the stiffness regime of the particle, the entire mechanism of uptake changes. Our interest for this report is to explore the potential for mechanosensitive endocytosis in a distinctly unique regime of particle stiffness. All previous studies of the particle mechanics and cell uptake used particles with Young’s moduli ranging from 15 kPa to 20 MPa. In this 2 ACS Paragon Plus Environment

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study, a series of poly(styrene-N-isopropylacrylamide) (pS-co-NIPAM) microgels were prepared over a range of stiffness values in the high MPa range, while controlling against particle size and charge, to directly evaluate and compare uptake efficiency in a phagocytic and nonphagocytic cell line: RAW264.7 macrophage and HepG2 hepatoma cells, respectively. Both of these cell lines have previously demonstrated mechanosensitive endocytosis6, 10, yet a direct comparison between RAW 264.7 and HepG2 mechanosensitive uptake is obscured by differences in particle size, composition, and surface chemistry. Overall, demonstration of mechanosensitive uptake in a new stiffness regime will expand potential material selection beyond microgels to further support particle mechanics in the design of novel drug-delivery vehicles. Furthermore, understanding the basic cell-particle dynamics that enable mechanosensitive endocytosis will enable fine tuning specific ranges of particle mechanics for optimization to increase tissue targeting and/or avoid of rapid clearance. 2. EXPERIMENTAL 2.1 Microgel synthesis. All chemicals were purchased from Sigma-Aldrich and used as received, unless otherwise stated. The particles were synthesized as done previously14. Briefly, Nisopropylacrylamide (NIPAM) and N, N’-methylenebisacrylamide (BIS) were dissolved in 100mL of nanopure water in a three-necked, round-bottom flask equipped with a water jacketed condenser, heated to 75°C, and purged with nitrogen gas. Styrene was added, and the reaction was initiated with the addition of potassium persulfate (KPS) (1mL, 75mg/mL). The reaction was allowed to run for 24 hours with constant stirring, followed by filtration through glass wool in order to remove any debris. Finally, unreacted NIPAM and KPS were removed through multiple centrifugation and redispersion steps in nanopure water. Polymer concentrations were maintained at 300mM for all syntheses. For quantifying and imaging pS-co-NIPAM uptake, a fluorescent tag (methacryloxyethyl thiocarbonyl rhodamine B, PolyFluor 570) was added (0.005 mg/mL) to the reaction vessel prior to the addition of styrene. All other reactant concentrations remained the same. Incorporating the fluorescent tag into the co-polymer limits concern of potential tag leaching, and, for our uptake studies, the measured fluorescence is correlated to the number of particles either bound or internalized by RAW264.7 or HepG2 cells. 2.2 Microgel size characterization: DLS and AFM imaging. Air-dried samples of fluorescently tagged pS-co-NIPAM were imaged using Atomic Force Microscopy (AFM) AC mode imaging, as done previously14. Briefly, Si wafers (Ted Pella Inc.) were cleaned by sonicating in ethanol for 5 minutes, rinsed with water, then ethanol, and air dried. Dilute suspensions of the pS-co-NIPAM microgels were then drop-cast onto the wafers. All the AFM measurements were performed using a Molecular Force Probe 3D AFM (Asylum Research). Reported height and width values of microgels were an average of ~50 individual particles that were obtained using a cross-sectional analysis from AFM images. For determining the microgel size in cell culture media (serum-free DMEM), dynamic light scattering (DLS) was used for complementary size assessment and to identify potential particle aggregation. The laser wavelength (λ = 633 nm) used for our DLS studies was outside the excitation band for our tagged pS-co-NIPAM particles, so potential fluorescence did not interfere with the scattering-based sizing studies. Dilute microgel suspensions were prepared in media and characterized using a Zetasizer Nano-ZS (Malvern Instruments) using a 173o backscattering geometry. The microgel particles were sized in a liquid cell equilibrated at 37 oC for approximately 10 minutes prior to measurement. The particle size was determined by DLS in triplicate measurements; our results are reported as the average, with standard deviation representing the error. 3 ACS Paragon Plus Environment


2.3 Microgel mechanics and AFM nanoindentation. To determine the mechanical properties of hydrated microgels at 37 oC, pS-co-NIPAM microgels were first immobilized onto a glass substrate following a modified approach reported by Xia et al.15-16 Briefly, glass coverslips (Asylum Research) were immersed in a freshly prepared piranha solution (3:1 mixture of 98% H2SO4 and 30% H2O2) at 100 oC for one hour to a clean, negatively charged glass surface. These coverslips were submerged in a 1 wt% polyethylene imine (PEI) solution, followed by a thorough washing with deionized water and drying under a flow of nitrogen gas. A monolayer of the pS-co-NIPAM sample was deposited using a spin coater (Laurell Technologies Corp.) onto the PEI-coated glass coverslips with a 150 µL of a 1 wt% microgel suspension. Spin coating was performed at 2000 rpm for 2 minutes. The coverslips with deposited microgel sample were annealed in a water bath (~ 65 o C) for 12 hrs while replacing the water solution at least three times. Note that the deposition was poor for the tagged pS-co-NIPAM particles, thus the mechanical measurements reported are for the untagged particles. The untagged particles were prepared using the same protocol described above only without the addition of the fluorophore. All nanoindentation and imaging experiments were performed as reported in our previous work on the mechanical properties of air-dried pS-co-NIPAM microgels.14 Briefly, AFM measurements were performed in a liquid cell at 37 ± 0.1 oC. Four different Si3N4 probes (Mikromasch), with a spring constant range of 0.2 N/m, were used for particle imaging and force spectroscopy. Actual spring constants were measured before every nanoindentation experiment using a built-in thermal noise method17. In order to locate individual pS-co-NIPAM microgels for the nanoindentation experiment, AFM AC mode imaging was performed with a slower scan rate of 0.5 Hz under liquid environment to minimize the particle detachment from the surface in liquid. The nanoindentation experiments were performed over an approximate center of an individual microgel particle in the contact mode with the applied force range from 5 – 8 nN, and 500 nms-1 approach velocity.18-20 Johnson-Kendall-Roberts (JKR) model was utilized to calculate the stiffness (Young’s modulus) values of hydrated pS-co-NIPAM particles by fitting the approach data collected during nanoindentation experiments.21 Manufacturer provided 10 nm tip radius of curvature and a Poisson’s ratio of 0.25 were used to determine the stiffness values of hydrated pS-co-NIPAM particles with JKR model. Stiffness values obtained from the analysis are an average of three consecutive force measurements collected on top of a single particle with a minimum of 15 different particles studied for each pS-co-NIPAM composition. 2.4 Cell culture and viability. RAW264.7 macrophages were purchased from American Type Culture Collection (ATCC) and grown in complete medium consisting of Dulbecco’s Modified Eagle culture medium (DMEM) containing 10% (vol/vol) fetal calf serum (FCS), 10mM HEPES (Invitrogen), 1mM sodium pyruvate (Invitrogen), 1% penicillin-G and streptomycin (Invitrogen) in 5% CO2 atmosphere at 37°C. All cell studies in this report used cells at passage numbers between 5 – 15. HepG2 human hepatoma cells were a gift from Prof. Frederick Domann (University of Iowa) and maintained in a T75 flask, passaged every three days with a sub-culturing density of 1:4. HepG2 cells were cultured in a low-glucose DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1% glutamax (Gibco) and 1% penicillin-G and streptomycin. All cell studies in this report used cells at passage numbers between 20 – 25. After incubation with our pS-co-NIPAM microgels, cell viability was determined using the MTS assay. This method assesses cell viability by analyzing the reduction of a yellow tetrazolium component, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium), to a purple formazan product.22 RAW264.7 cells were plated at a seeding density of 1x104 cells/well in flat-bottom 96-well plates and cultured for 48 hours in complete growth medium. The media was removed, and the cells were incubated with pS-co4 ACS Paragon Plus Environment

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NIPAM particles in serum-free DMEM for 24 hrs. Cells incubated in serum-free media without pS-co-NIPAM particles served as the control. The particle suspension was removed and replaced with fresh DMEM containing MTS. The plate was further incubated at 37°C for 1 hour for color development. The color intensity of the formazan product was then measured by analyzing the absorbance at 490nm. The same protocol was followed for assessing viability of the HepG2 cells upon exposure to our pS-co-NIPAM microgels with a seeding density of 2x104 cells per well and serum-free RPMI-1640 supplemented with 1% penicillin-streptomycin. All reported values are normalized to the absorbance value provided from the control well, which is assumed to represent 100% viability. Error bars represent the standard deviation All viability studies were analyzed using a one-way ANOVA (n=6 in triplicate). 2.5 Microgel endocytosis and mechanism discrimination. RAW264.7 cells were plated in white 96-well flat bottom plates (1 x 104 cells/well) and grown for 48 hours in complete growth medium. The growth medium was removed and replaced with serum-free DMEM containing 90 µg/mL of the fluorescent microgel suspensions. After various time points (2-24hrs), the media was removed, and the cells were washed three times with Dulbecco’s phosphate buffered saline (DPBS). The cells were lysed with a 10mM potassium phosphate buffer containing 0.1% Triton-X (pH = 7.5). The plate was placed on a shaker table for 10 minutes at 120rpm, and the fluorescence of the lysate was measured using a SpectraMax M5 (Molecular Devices) plate reader with excitation and emission wavelengths at 520 and 575nm,respectively . This same protocol was followed for determining microgel uptake into the HepG2 cells, again using a seeding density of 2x104 cells per well and serum-free RPMI-1640. Our calibration curve was prepared by serial dilution of each pS-co-NIPAM suspension prepared in lysis buffer (n=12 in triplicate). For identifying the participation of specific uptake modes, a series of pharmacological inhibitors were used. Prior to particle incubation, MTS assays confirmed that our inhibitor doses did not significantly diminish cell viability during the 5 hour duration of our study. This permits a direct comparison of each inhibitor to identify a potentially preferred mode of particle endocytosis. The inhibitors and optimized dosing concentrations for RAW264.7 cells were as follows: chlorpromazine (20 µg/mL), methyl-β-cyclodextrin (5 mM), nystatin (50 U/mL), amiloride (200 µg/mL), cytochalasin D (5 µg/mL), genistein (37 µM), and nocodazole (5 µg/mL). For HepG2 our inhibitor doses were the same for chlorpromazine, methyl-β-cyclodextrin, cytochalasin D, and genistein. Higher doses of nystatin (200 µg/mL), amiloride (1 mM), and nocodazole (1 mM) were used with the HepG2 cells in accordance with the literature23-24. Each inhibitor was added to the cells and incubated at 37°C with 5% CO2 for 1 hour. The inhibitor solution was then removed and replaced with microgels suspended in serum- and phenol-free media. The amount of uptake was measured after 4 hours using lysate fluorescence, and our results are reported as a percent control of the untreated sample. All uptake and kinetic analyses were done using a two-way ANOVA, with a Tukey post hoc test (n=8 in triplicate). 2.6 Confocal fluorescence Complementary studies of particle uptake into both cell lines were performed using confocal immunofluorescence. RAW264.7 and HepG2 cells were separately plated on 18mm glass coverslips in 12-well plates (2.5 x 105 cells/well) and grown in full media for 48 hours. The cells were incubated with a 90 µg/mL microgel suspension in serum and phenol-free media for four hours. The cells were then washed three times in ice cold DPBS and fixed in ice cold 4% paraformaldehyde for 10 minutes. The cells were washed in DPBS, and the membrane was permeated with 0.1% Triton-X for 5 minutes. The cells were then stained with 0.165 µM of AlexaFluor488-phalloidin to tag for F-actin. Finally the coverslips were removed from the well and placed on a microscope slide with 1 drop of Vectashield® containing DAPI as a nuclear stain. Slides were imaged using a Zeiss LSM710 confocal microscope. 5 ACS Paragon Plus Environment


3. RESULTS AND DISCUSSION 3.1 Microgel characterization: Size and particle mechanics Polymeric composition, size, zeta potential and mechanical data for our pS-co-NIPAM series are collected for reference in Table I. For our microgel series, all compositions yielded microgels with diameter between 300-470 nm. Selected AFM images, as shown in Figure 1, demonstrate our microgels all display a spherical particle morphology and relatively low polydispersity. Some particle flattening is observed for the air-dried pS-co-NIPAM particles with measured widths being systematically larger than the particle heights. These AFM results are consistent with our DLS studies of microgel size in media and at 37 oC. Some sample-tosample variation in particle diameter is observed with pS-co-NIPAM-2 being the smallest (D = 305 nm) and pS-co-NIPAM-3 the largest (D = 464 nm). Furthermore, some particle aggregation is observed for pS-co-NIPAM-1 suspensions in cell media displaying the highest polydispersity index at 0.31 for the microgel series. All microgels suspended in cell media at 37 oC display a slight negative charge ranging from approximately -8 to -11 mV. No significant difference in the zeta potential was observed between the three particles. AFM nanoindentation was performed to assess the mechanical properties of our hydrated pS-co-NIPAM microgel series at 37 oC and all force-displacement curves, with fits to the JKR model, are provided in Figure 2. As illustrated in Figure 2, multiple, individual microgels were sampled to provide a robust data set for determining the mechanical properties. Despite some microgel-to-microgel variability, the modulus obtained from the JKR fit should be considered an average Young’s modulus representative of the microgel distribution. Further details of this analysis are provided as Supporting Information. Note that for our analysis, we used the Poisson’s ratio previously determined for the air-dried pS-co-NIPAM particles using Brillouin light scattering14 and the final outcome of this analysis are provided in Figure 3. The general expectation is an increasing crosslinker concentration results in an increased microgel stiffness, which is consistent with our measured mechanical properties of our pS-co-NIPAM series. Our pS-co-NIPAM microgels demonstrate mid-range Young’s moduli relative to either of the pure polymers, and as expected from our previous mechanical studies of air-dried pS-coNIPAM, the introduction of styrene increases microgel stiffness significantly relative to pure NIPAM25. Our softest microgel, pS-co-NIPAM-1 has an E = 310 MPa, while pS-co-NIPAM-3 yielded the stiffest material with an E = 720 MPa, showing that an increase in the crosslinking concentration directly increases the stiffness of the microgel The immobilization of these pS-coNIPAM microgels onto a substrate for in-liquid measurement of particle mechanics was challenging, particularly for the rhodamine-labeled pS-co-NIPAM particles. Therefore, the Young’s moduli provided in Table I are for the untagged pS-co-NIPAM particles which were prepared following an identical protocol, without the addition of the fluorescent tag. Our expectation is that any introduced mechanical variation, if any, would be consistent across the series since the concentration of the fluorophore is the same across all microgels, and the relative differences in Young’s moduli would remain valid. To confirm this expectation, an air-dried sample of the tagged pS-co-NIPAM was measured and compared with our previous work. The results of this comparison are provided in the Supporting Information (Figure S1), and demonstrate minor variation between the mechanical properties of untagged and tagged pS-co-NIPAM microgels. Thus we consider our mechanical data for the hydrated, untagged pS-co-NIPAM as a suitable representative of the tagged microgel mechanics used in our uptake studies. This enables exploring potential mechanosensitive endocytosis in a new stiffness regime distinct from previous microgel uptake studies. Pure NIPAM displays a volume phase transition at approximately 32 oC whereby as temperature increases through this transition, water is expelled from the microgel interior accompanied by particle collapse and a significant reduction in volume. As demonstrated by 6 ACS Paragon Plus Environment

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Tagit et al., the mechanical properties of swollen and collapsed pNIPAM are significantly different, with the Young’s modulus of the collapsed particle (E = 12.8 MPa) nearly an order of magnitude larger than the water-swollen particle (E = 1.8 MPa).25 Through co-polymerizing styrene, a hydrophobic monomer, with NIPAM the temperature of volume phase transition reduces as does the extent of the particle volume reduction. At high styrene incorporation (≥ 50 mol%), no volume transition occurs and our previous study has confirmed this expectation for pS-co-NIPAM-1 through -3 from 10 – 50 oC.14, 26-27 This is further consistent with our mechanical data for the pS-co-NIPAM microgels in water showing essentially no difference from our previous work on air-dried particles14. 3.2 Cell viability NIPAM-based microgels of varying composition have routinely displayed low cytotoxicity with excellent biocompatibility, however, unreacted N-isopropylacrylamide and bis-acrylamide monomer have displayed significant cytotoxicity across multiple cell lines.28-31 For pS-coNIPAM, no previous cytotoxicity results are available, thus the relative cell viability of both HepG2 and RAW264.7 cells incubated with our microgel series was evaluated using an MTS assay. Our cell viability with MTS assesses the metabolic activity of the cell, which is proportional to the absorbance of the reduced formazan product at 490 nm. To maintain this proportionality, seeding densities for both cell lines were within the manufacturer’s recommendations to avoid saturating absorbance detection. As shown in Figure 4, for our highest dosing concentration (150 µg/mL) with an exposure period of 24 hours, neither HepG2 nor RAW264.7 cells display any reduction in viability upon microgel exposure. This permits a direct comparison of particle uptake without confounding concerns of cytotoxicity. Eliminating this concern of potential cytotoxicity guided our range of doses rather than any specific therapeutic dose. 3.3 Cell-specific mechanosensitive uptake: Dose-response and kinetics Particle mechanics have previously been demonstrated to be impactful in multiple in vitro cell uptake and in vivo biodistribution studies. Collectively, however, these studies provide no consensus from a particle design perspective of whether particle stiffening or softening is advantageous for a specific application or a specific cell-line. For example, Banquy et al. had previously demonstrated mechanosensitive endocytosis for RAW264.7 internalization of N,N′diethylacrylamide-co-2-hydroxyethylmethacryalte microgels.8 These microgels were approximately half the diameter of our particles and in a completely separate stiffness regime with Young’s moduli spanning 18 – 211 kPa. For those studies, the softest microgels consistently displayed the lowest particle uptake, while mid-range stiffness microgels were internalized at the greatest extent over a 4 hr incubation period. The expectation that soft particles may frustrate internalization was further corroborated by a theoretical model reported by Yi et al.11 While greater particle deformation and a low particle stiffness (relative to the bending modulus of the cell membrane) promotes adhesion, the energetic cost of membrane wrapping a deformed particle suggests there would be a reduction in particle uptake. However, the study reported by Liu et al. for large crosslinked pHEMA particles with diameter consistently near one micron and Young’s moduli ranging from 17 – 156 kPa displayed the highest uptake efficiency for the softest microgels in HepG2 cells13. There was a stark difference in uptake between the two softer microgels and the two stiffer microgels as a function of both time and dose; which corresponds well with the decreased uptake observed for the stiffest particles in the present study13. Cell-specific mechanosensitive endocytosis is expected, however, further controlled studies are needed to identify how alternative cell lines integrate particle mechanics into an endocytic mechanism. For our comparative uptake studies with RAW264.7 and HepG2 cells, both the doseresponse and kinetics of pS-co-NIPAM uptake were quantitatively evaluated using lysate 7 ACS Paragon Plus Environment


fluorescence and these results are respectively shown in Figures 5 and 6 for each cell line. At minimum, three PBS rinses were applied prior to cell lysis and fluorescence measurement to remove non-internalized and loosely adhered microgels. Confocal images were also captured to help confirm microgel internalization. However, despite this we note that our fluorescencebased measurement of uptake concentration may contain both internalized and strongly adhered microgels. For both cell lines and all pS-co-NIPAM compositions, with an escalating dose the overall uptake concentration increased. Notably for both cell lines, even in our highstiffness regime, particle mechanics clearly influences both the dose-response and the kinetics of microgel uptake. For RAW 264.7 cells, after a four hour incubation, the softest microgel of our series, pS-co-NIPAM-1, was endocytosed significantly faster than the other two microgels. This order of uptake efficiency, at all microgel concentrations, is pS-co-NIPAM-1 > pS-coNIPAM-2 > pS-co-NIPAM-3, which mirrors a clear trend in particle mechanics from softest to stiffest, respectively. The differences in uptake concentration between separate microgels becomes significantly more pronounced in a dose-dependent fashion. At our highest microgel dose (150 µg/mL), the internalized pS-co-NIPAM-1 concentration displayed a five-fold increase relative to pS-co-NIPAM-3, the stiffest particle (E = 720 MPa). However, pS-co-NIPAM-2 was also significantly smaller than the other two microgels in the series. As size is known to play a significant role in the endocytosis of particles, the increased size of pS-co-NIPAM-3 may have further limited uptake. However, if size were the dominant factor driving microgel uptake, then pS-co-NIPAM-2 should have displayed the highest uptake, as it was the smallest of the three microgels. Overall, the expectation is particle mechanics is the primary discriminant of our observed differences in internalization efficiency. For HepG2 cells, the concentration dependence of microgel uptake shown in Figure 6 is distinct from our uptake studies using RAW264.7, indicating that mechanosensitive endocytosis is a cell-specific response. Overall uptake is considerably reduced relative to that observed for the RAW264.7 macrophage. The uptake concentration does increase in a dose-dependent manner; however, no significant influence of particle mechanics is observed between pS-coNIPAM-1 and pS-co-NIPAM-2 from 15 – 90 µg/mL doses under a 4 hour incubation. However, at all dosing concentrations, both of these microgels, display a significant increase in uptake relative to the stiffest particle, pS-co-NIPAM-3. Only at the highest dose (150 µg/mL) is a clear increase in pS-co-NIPAM-1 observed relative to the other microgels. The dose-dependent uptake was similarly observed in HepG2. At all dose concentrations, pS-co-NIPAM-3 has a significantly decreased uptake, while a mechanosensitive difference is not observed between pS-co-NIPAM-1 and 2 until the highest dose. Cell-specific uptake behavior further discriminates the kinetics of pS-co-NIPAM internalization. For RAW264.7 cells dosed at 90 µg/mL, softer pS-co-NIPAM particles (both pSco-NIPAM-1 and pS-co-NIPAM-2) were internalized at a faster rate relative to pS-co-NIPAM-3, most notably at our 4, 8 and 12 hour time points. However, after a 24 hour incubation period, all particles reach approximately the same uptake concentration, potentially due to uptake saturation. Similar results were observed for HepG2 cells with the softest microgel displaying faster internalization of the softer particles, most notably beginning at the 8 hour time point, however, after a 24 hour incubation the softest microgel maintained a marginal increase in uptake, where no uptake saturation was observed. These lysate studies for both RAW264.7 and HepG2 cells are qualitatively consistent with our confocal fluorescence images displayed in Figures 7 and 8. At a 90 µg/mL dose, only modest fluorescence is observed for pS-co-NIPAM-3 in both cell lines, indicating limited uptake of this microgel. With particle softening, the fluorescence increases to a maximum observed for the pS-co-NIPAM-1. Further seen in Figure 7 and 8 is the intracellular accumulation of the microgels; the primary fraction of pS-co-NIPAM particles are not simply adhered to the cell membrane, but rather internalized, presumably through an energy-dependent process. Particle 8 ACS Paragon Plus Environment

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mechanics, even at extremely high-stiffness’, can dramatically adjust internalization of particles, especially in phagocytic cells such as RAW264.7. Our observed kinetics of microgel uptake agrees well with the theoretical model reported by Yi et al. for the of uptake kinetics of elastically deformable particles32. Critical elements of their model include particle stiffness and size, cell-membrane tension and binding strength at the particle-membrane interface. This model demonstrates that soft particles are less energetically prone to wrapping relative to stiff particles. However, once wrapping becomes energetically favorable, softer particles exhibit an increased uptake compared to stiff particles. For our results, after a two-hour incubation only minor uptake was observed in RAW264.7 cells; however after four hours microgel mechanosensitivity dramatically increased, showing increased uptake, most notably for the softer microgels, pS-co-NIPAM-1 and pS-co-NIPAM-2. For the stiffest microgel, substantial uptake (at 90 µg/mL dose) did not occur until near eight hours. Similar results are observed for the uptake kinetics in HepG2 cells; however, less than one tenth of the total particle uptake concentration was achieved in HepG2 cells compared with the RAW264.7 macrophages. This is a common trend observed in the literature when comparing phagocytic and non-phagocytic cells33-35. The improved kinetics of soft particle internalization is facilitated by higher contact area between the membrane and the particle at early stages of wrapping. Since a softer particle can be more easily deformed during the early stages of membrane wrapping, there is a higher percentage of cells bound with soft particles, than cells bound with stiff particles during the early incubation period. This interaction between the cell and particle is sensitive to the microgel surface chemistry. Because it has been shown that a protein corona can affect particle uptake,36 serum-free media was used in all our studies. Moreover, two previous studies by Beningo et al. and Liu et al. demonstrate that particles with mechanical stiffness adjusted through varying the crosslinking density displayed no differences in protein adsorption7, 13Thus, for our in vitro experiments we do not expect differential protein adsorption to play a significant role in the uptake observed for our pS-co-NIPAM series. These pS-co-NIPAM microgels are distinct, as they neither display a homogenous composition, nor do they have a sharp core-shell structure. While both KPA and BIS act as crosslinking agents for pNIPAM, each material differs in the region of crosslinking. KPS acts to crosslink the pNIPAM at the periphery of the particle, while BIS act more predominantly at the particle’s core. At low concentrations of BIS crosslinking, NIPAM polymerization is initiated at the particle surface and gradually moves toward the particle core37. This leaves the interior of the microgel as predominantly polystyrene, while the exterior remains mostly crosslinked pNIPAM, with a gradient occurring from the interior core of the particle outward. This crosslinking suggests that pS-co-NIPAM-3, with the highest amount of BIS, has a lower crosslinking density at the particle surface. This difference of crosslinking at the particle surface, along with particle mechanics, potentially contribute to particle uptake discrimination. Our pS-co-NIPAM particles are not ligand modified, nor any protein corona formation is expected in our serum-free media, thus we assume only a non-specific interaction exists at the particle-membrane interface for either the RAW264.7 or HepG2 uptake studies. These nonspecific interactions at the particle membrane are important to endocytosis, as demonstrated by Decuzzi et al., and thus cell-specific differences in both membrane tension and non-specific particle-membrane interactions may underscore their differences in pS-co-NIPAM internalization.38 Overall, the physical properties of both the particle and the cell are important governing factors in particle recognition at the cell membrane and subsequent endocytosis. 3.4 Pharmacological inhibitors and endocytic mechanism The cell membrane strictly regulates particle entry with phagocytosis and a variety of energy-dependent pathways for particle endocytosis such as macropinocytosis, caveolaedependent, and clathrin-mediated uptake.39-42 To identify whether energy-dependent processes 9 ACS Paragon Plus Environment


are active for pS-co-NIPAM internalization, our uptake studies were repeated at 4 oC for both RAW264.7 and HepG2. Using this cold culture, all energy-dependent processes are stopped or significantly slowed and if energy-dependent pathways are operative then a significant reduction in uptake concentration would be observed relative to the control experiments performed at 37oC. Moreover, as mentioned previously, our pS-co-NIPAM microgels display no swelling behavior, thus using a cold culture introduces no concern about changes in particle sizes. Pure pNIPAM exhibits volume phase transition at near 32 oC whereby heating through this transition temperature results in microgel collapse. This collapse is typically argued to result from a change in hydrophilicity, however, recent measurements reported by Zhuang et al. demonstrate that no hydrophilic-hydrophobic transition accompanies the volume phase transition. For our pS-co-NIPAM microgels, no volume phase transition is observed due the incorporation of a high mol% of styrene. Thus, the expectedly minor changes in pS-co-NIPAM size and wettability at 4 oC permits the use of a cold culture to identify energy-dependent uptake processes. The results of this cold culture are shown in Figure 9, and clearly identify pS-co-NIPAM internalization in either cell line follows an energy-dependent process. Most notably for the RAW264.7 cells, this large difference in uptake concentration helps further support the expectation that the primary contribution to the fluorescence measurement is from internalized particles. There was, however, no significant difference for the pS-co-NIPAM-3 between the two temperatures in the HepG2. This could be a consequence of the low uptake for that microgel, or could suggest the uptake of the stiffest particle is not necessarily energydependent. It is likely that given the significant differences between RAW264.7 and HepG2 in terms of particle uptake efficiency and kinetics these cell lines internalize pS-co-NIPAM microgels through distinctly separate routes. The mechanosensitive, differential activation of separate endocytic routes has previously been demonstrated by Banquy et al. for RAW 264.7 cells.8 Soft and stiff nanoparticles were preferentially internalized through macropinocytic and clathrin-mediated uptake, respectively, as determined through systematic pre-treatment of cells with specific endocytic inhibitors. Those microgels with mid-range elasticity, however, activated multiple entry mechanisms and consequently displayed a larger, overall uptake efficiency. A variety of pharmacological inhibitors are available to assess potentially preferential modes of endocytosis.43-46 For our studies we chose chlorpromazine, methyl-β-cyclodextrin, nystatin, amiloride, cytochalasin D, genistein, and nocodazole. With higher inhibitor concentrations, Liu, et al. observed decreased uptake of pHEMA/BIS particles in HepG2 cells when the following mechanisms were inhibited: clathrin-mediated endocytosis, caveolae-mediated endocytosis, micropinocytosis, and energydependent endocytosis.10 However, our preliminary viability studies indicated that the HepG2 cells indicated that higher concentrations may be causing cell death, which could be a reason for decreased uptake. Above certain threshold concentrations inhibitors can be cytotoxic, and thus MTS studies were performed to ensure cell viability was not reduced using a one-hour pretreatment of the inhibitor followed by a 4 hr incubation with a 90 µg/mL pS-co-NIPAM dose. Following this protocol no reduction in cell viability was observed for either RAW264.7 or HepG2 cells. Uptake results for RAW264.7 and HepG2 cells pre-treated with all inhibitors are provided as Figure 10. The only inhibitors that displayed a significant difference relative to the control cells, i.e. same particle dose and incubation period but without inhibitor pre-treatment, are cytochalasin D and nocodazole for RAW264.7 only. Cytochalasin D is reported to bind with high affinity to the growing, barbed, end of F-actin filaments and prevents further polymerization with G-actin. Polymerization of actin is an important element of macropinocytosis and drives membrane ruffling and lamellipodia formation and extension. Some lamellipodia subsequently fold back and re-fuse with cell membrane engulfing extracellular contents into a macropinosome. This endocytic process is typically utilized for large particle intake (>200 nm) 10 ACS Paragon Plus Environment

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and thus appropriate for the internalization of our pS-co-NIPAM microgels. In contrast to the previous studies using RAW264.7, our studies find that the stiffest microgels (in the high MPa regime) are preferentially internalized through macropinocytosis. In contrast, the HepG2 cells did not display decreased uptake for any of the particle types in the presence of cytochalasin D (Figure 10), although some reduction in cell viability was observed following a 1 hour incubation with 5 µg/mL cytochalasin D and a 24 hour incubation in serum-free media (data not shown). These data are consistent with graphene oxide nanoparticle uptake in HepG2 cells, in which approximately half the dose of cytochalasin D was applied prior to nanoparticle exposure47. Nocodazole acts to inhibit microtubule polymerization through high affinity binding to tubulin and can directly modify phagocytosis. Phagocytosis involves a coordinated restructuring of the cell membrane with the actin cytoskeleton. Although the actin cytoskeleton is typically emphasized, the potential microtubule role is poorly understood. Over our range of particle mechanics, arresting microtubule polymerization using nocodazole reduced microgel internalization in a progressive, mechanics-dependent manner, with our softest particles displaying the highest reduction in uptake for nocodazole-treated RAW264.7 cells. Macrophages are competent phagocytes, and again consistent with our particle size range, phagocytosis would be an expected mode of particle entry. By contrasting the results of our inhibitor studies it is clear that macropinocytosis and phagocytosis, respond to particle mechanics in a distinct manner at our high-stiffness regime for phagocytic cells. Nocodazole did not however show a significant decrease in uptake for HepG2. Moreover, this distinction highlights the separate role of both the actin and microtubule cytoskeletons in phagocytic cells for internalizing mechanically distinct particles. Our studies using pharmacological inhibitors did show a significant decrease in uptake in HepG2, other than when ATP production was stopped by completing the study at 4°C. Nocodazole, cytochalasin D, and amiloride all showed a decrease in cell viability 24 hours after incubation with the inhibitor in HepG2, while there was no inhibition of uptake after 4 hours. Seeing a reduction in cell viability after 24 hours suggests that a higher inhibitor dose would lead to a decrease in cell viability in the duration of our study. Previous studies have also suggested that amiloride could induce reduced particle uptake in both RAW264.78 and HepG246. However, at comparable concentrations, for each cell line, no inhibition was observed. Further, for RAW264.7, a lack of inhibition of uptake with chlorpromazine, methyl-beta cyclodextrin, nystatin, and genistein are consistent with what is found in the literature for phagocytic cells and particles in our size range48. For particles with similar sizes to our microgels, it is expected that macropinocytosis and phagocytosis would be the most likely paths of energy dependent uptake in a phagocytic cell. 4. CONCLUSIONS The mechanical properties of nanoparticles offer a unique design space to tune the efficiency of drug delivery vectors. Unlike alternative physical properties (i.e. size, shape, charge, etc.), previous studies demonstrating the effect of particle mechanics on endocytosis and intracellular distribution have been limited to approximately 15 kPa – 20 MPa. Our interest in this study is whether particle stiffness, at a greater than an order-of-magnitude higher stiffness than previous studies, will continue to demonstrate mechanosensitive endocytosis. To this end, a mechanical series of pS-co-NIPAM microgels were prepared with a constant styrene:NIPAM molar ratio (50 mol% styrene and 50 mol% NIPAM) but increasing bisacrylamide crosslinking concentration. Our AFM nanoindentation studies confirm that both the introduction of styrene and increasing crosslinking concentration produces microgels in a unique stiffness regime ranging from 310 – 720 MPa. Despite these high stiffness values, microgel endocytosis in both RAW264.7 and HepG2 remains sensitive to changes in particle mechanics but in a cell-specific manner. For RAW264.7 cell, the internalization of our softest microgel (pS11 ACS Paragon Plus Environment


co-NIPAM-1, E = 310 MPa) outpaced all remaining microgels in a dose-dependent fashion. At our highest dose (150 µg/mL), uptake of pS-co-NIPAM-1 was increased by five-fold relative to our stiffest microgel, pS-co-NIPAM-3. Our kinetic assessment of uptake further demonstrated that softer particles displayed faster uptake relative to pS-co-NIPAM-3; however, after a 24 hr incubation all microgels reached the same uptake plateau. These results are consistent with the theoretical model reported by Yi et al. Moreover, using a cold culture the uptake of all microgels in both cell lines follows an energy-dependent pathway. To further detail the specific mode of endocytosis, cells were pre-treated with a series of pharmacological inhibitors. Our uptake results for RAW264.7 indicate that the stiffest microgel moderately preferred actindependent macropinocytosis, while internalization of our softer particles were more effective through a microtubule-dependent pathway that is likely phagocytosis. For HepG2 cells overall uptake all microgels was significantly diminished relative to the uptake observed in RAW264.7 cells. Our softest microgel again displayed the highest uptake of the series, however, this was observed only at our highest dose concentration. Moreover, similar uptake kinetics were observed with both softer particles internalized at a faster rate than pS-co-NIPAM-3; however, unlike our results for RAW264.7, internalization of our stiffest particle in HepG2 cells displayed only a marginal increase after 24 hours. Only cold culture incubation, and thereby inhibition of energy-dependent endocytosis, resulted in decreased particle uptake in HepG2 cells. Overall, through demonstration of mechanosensitive endocytosis at a new stiffness regime, will facilitate the inclusion of other materials, beyond microgels, to broaden the functionality of this novel particle design space. 5. ACKNOWLEDGEMENTS Research reported in this publication was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under award number R21EB021035. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Furthermore, this work utilized the Zeiss LSM710 confocal microscope in the University of Iowa Central Microscopy Research Facilities that was purchased with funding from the NIH SIG grant S10 RR022498. Supporting Information. A complete description of the AFM nanoindentation measurement and fitting analysis. A comparison of air-dried tagged and untagged particle mechanics.

6. REFERENCES 1. Parveen, S.; Misra, R.; Sahoo, S. K., Nanoparticles: A Boon To Drug Delivery, Therapeutics, Diagnostics And Imaging. Nanomed-Nanotechnol 2012, 8 (2), 147-166. 2. Blanco, E.; Shen, H.; Ferrari, M., Principles Of Nanoparticle Design For Overcoming Biological Barriers To Drug Delivery. Nat Biotechnol 2015, 33 (9), 941-951. 3. Mitragotri, S.; Lahann, J., Physical Approaches To Biomaterial Design. Nat Mater 2009, 8 (1), 1523. 4. Mitragotri, S., In Drug Delivery, Shape Does Matter. Pharm Res 2009, 26 (1), 232-234. 5. Champion, J. A.; Katare, Y. K.; Mitragotri, S., Particle Shape: A New Design Parameter For MicroAnd Nanoscale Drug Delivery Carriers. J Control Release 2007, 121 (1), 3-9. 6. Zhang, S.; Gao, H.; Bao, G., Physical Principles Of Nanoparticle Cellular Endocytosis. ACS Nano 2015, 9 (9), 8655-8671. 12 ACS Paragon Plus Environment

Page 12 of 25

Page 13 of 25 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


7. Beningo, K. A.; Wang, Y.-L., Fc-Receptor-Mediated Phagocytosis Is Regulated By Mechanical Properties Of The Target. J Cell Sci 2002, 115 (4), 849-856. 8. Banquy, X.; Suarez, F.; Argaw, A.; Rabanel, J.-M.; Grutter, P.; Bouchard, J.-F.; Hildgen, P.; Giasson, S., Effect Of Mechanical Properties Of Hydrogel Nanoparticles On Macrophage Cell Uptake. Soft Matter 2009, 5 (20), 3984-3991. 9. Anselmo, A. C.; Mitragotri, S., Impact Of Particle Elasticity On Particle-Based Drug Delivery Systems. Adv Drug Deliver Rev 2017, 108, 51-67. 10. Sun, H.; Wong, E. H.; Yan, Y.; Cui, J.; Dai, Q.; Guo, J.; Qiao, G. G.; Caruso, F., The Role Of Capsule Stiffness On Cellular Processing. Chem Sci 2015, 6 (6), 3505-3514. 11. Yi, X.; Shi, X.; Gao, H., Cellular Uptake Of Elastic Nanoparticles. Phys Rev Lett 2011, 107 (9), 098101. 12. Anselmo, A. C.; Zhang, M.; Kumar, S.; Vogus, D. R.; Menegatti, S.; Helgeson, M. E.; Mitragotri, S., Elasticity Of Nanoparticles Influences Their Blood Circulation, Phagocytosis, Endocytosis, And Targeting. ACS Nano 2015, 9 (3), 3169-3177. 13. Liu, W.; Zhou, X.; Mao, Z.; Yu, D.; Wang, B.; Gao, C., Uptake Of Hydrogel Particles With Different Stiffness And Its Influence On Hepg2 Cell Functions. Soft Matter 2012, 8 (35), 9235-9245. 14. Mohapatra, H.; Kruger, T. M.; Lansakara, T. I.; Tivanski, A. V.; Stevens, L. L., Core And Surface Microgel Mechanics Are Differentially Sensitive To Alternative Crosslinking Concentrations. Soft Matter 2017, 13 (34), 5684-5695. 15. Xia, Y.; Tang, Y.; He, X.; Pan, F.; Li, Z.; Xu, H.; Lu, J. R., Patterned Thermoresponsive Microgel Surfaces To Control Cell Detachment. Biomacromolecules 2016, 17 (2), 572-579. 16. Xia, Y.; Tang, Y.; Wu, H.; Zhang, J.; Li, Z.; Pan, F.; Wang, S.; Wang, X.; Xu, H.; Lu, J. R., Fabrication Of Patterned Thermoresponsive Microgel Strips On Cell-Adherent Background And Their Application For Cell Sheet Recovery. ACS Appl Mater Inter 2017, 9 (2), 1255-1262. 17. Hutter, J. L.; Bechhoefer, J., Calibration Of Atomic-Force Microscope Tips. Rev Sci Instrum 1993, 64 (7), 1868-1873. 18. Peter, K. T.; Vargo, J. D.; Rupasinghe, T. P.; De Jesus, A.; Tivanski, A. V.; Sander, E. A.; Myung, N. V.; Cwiertny, D. M., Synthesis, Optimization, And Performance Demonstration Of Electrospun Carbon Nanofiber–Carbon Nanotube Composite Sorbents For Point-Of-Use Water Treatment. ACS Appl Mater Inter 2016, 8 (18), 11431-11440. 19. Rupasinghe, T. P.; Hutchins, K. M.; Bandaranayake, B. S.; Ghorai, S.; Karunatilake, C.; BučAr, D.-K. I.; Swenson, D. C.; Arnold, M. A.; Macgillivray, L. R.; Tivanski, A. V., Mechanical Properties Of A Series Of Macro-And Nanodimensional Organic Cocrystals Correlate With Atomic Polarizability. J Am Chem Soc 2015, 137 (40), 12768-12771. 20. Lee, H. D.; Ray, K. K.; Tivanski, A. V., Solid, Semisolid, And Liquid Phase States Of Individual Submicrometer Particles Directly Probed Using Atomic Force Microscopy. Anal Bioanal Chem 2017, 89 (23), 12720-12726. 21. Johnson, K.; Kendall, K.; Roberts, A. In Surface Energy And The Contact Of Elastic Solids, Proceedings Of The Royal Society Of London Series A, The Royal Society: 1971; Pp 301-313. 22. Goodwin, C.; Holt, S.; Downes, S.; Marshall, N., Microculture Tetrazolium Assays: A Comparison Between Two New Tetrazolium Salts, XTT And MTS. J Immunol Methods 1995, 179 (1), 95-103. 23. He, J.; Zheng, Y. W.; Lin, Y. F.; Mi, S.; Qin, X. W.; Weng, S. P.; He, J. G.; Guo, C. J., Caveolae Restrict Tiger Frog Virus Release In Hepg2 Cells And Caveolae-Associated Proteins Incorporated Into Virus Particles. Sci Rep 2016, 6, 21663. 24. Narva, S.; Chitti, S.; Amaroju, S.; Bhattacharjee, D.; Rao, B. B.; Jain, N.; Alvala, M.; Sekhar, K., Design And Synthesis Of 4-Morpholino-6-(1,2,3,6-Tetrahydropyridin-4-Yl)-N-(3,4,5-Trimethoxyphenyl)1,3,5- Triazin-2-Amine Analogues As Tubulin Polymerization Inhibitors. Bioorg Med Chem Lett 2017, 27 (16), 3794-3801. 13 ACS Paragon Plus Environment


25. Tagit, O.; Tomczak, N.; Vancso, G. J., Probing The Morphology And Nanoscale Mechanics Of Single Poly (N-Isopropylacrylamide) Microgels Across The Lower-Critical-Solution Temperature By Atomic Force Microscopy. Small 2008, 4 (1), 119-126. 26. Hellweg, T.; Dewhurst, C. D.; Eimer, W.; Kratz, K., PNIPAM-Co-Polystyrene Core− Shell Microgels: Structure, Swelling Behavior, And Crystallization. Langmuir 2004, 20 (11), 4330-4335. 27. Mcgrath, J. G.; Bock, R. D.; Cathcart, J. M.; Lyon, L. A., Self-Assembly Of “Paint-On” Colloidal Crystals Using Poly (Styrene-Co-N-Isopropylacrylamide) Spheres. Chem Mater 2007, 19 (7), 1584-1591. 28. Cooperstein, M. A.; Canavan, H. E., Assessment Of Cytotoxicity Of (N-Isopropyl Acrylamide) And Poly (N-Isopropyl Acrylamide)-Coated Surfaces. Biointerphases 2013, 8 (1), 19. 29. Edwards, P. M., Neurotoxicity Of Acrylamide And Its Analogues And Effects Of These Analogues And Other Agents On Acrylamide Neuropathy. Occ Environ Med 1975, 32 (1), 31-38. 30. Das, D.; Ghosh, P.; Ghosh, A.; Haldar, C.; Dhara, S.; Panda, A. B.; Pal, S., Stimulus-Responsive, Biodegradable, Biocompatible, Covalently Cross-Linked Hydrogel Based On Dextrin And Poly (NIsopropylacrylamide) For In Vitro/In Vivo Controlled Drug Release. ACS Appl Mater Inter 2015, 7 (26), 14338-14351. 31. Guo, Z.; Li, S.; Wang, C.; Xu, J.; Kirk, B.; Wu, J.; Liu, Z.; Xue, W., Biocompatibility And Cellular Uptake Mechanisms Of Poly (N-Isopropylacrylamide) In Different Cells. J Bioact Compat Pol 2017, 32 (1), 17-31. 32. Yi, X.; Gao, H., Kinetics Of Receptor-Mediated Endocytosis Of Elastic Nanoparticles. Nanoscale 2017, 9 (1), 454-463. 33. Liu, X.; Huang, N.; Li, H.; Jin, Q.; Ji, J., Surface And Size Effects On Cell Interaction Of Gold Nanoparticles With Both Phagocytic And Nonphagocytic Cells. Langmuir 2013, 29 (29), 9138-9148. 34. Lankoff, A.; Sandberg, W. J.; Wegierek-Ciuk, A.; Lisowska, H.; Refsnes, M.; Sartowska, B.; Schwarze, P. E.; Meczynska-Wielgosz, S.; Wojewodzka, M.; Kruszewski, M., The Effect Of Agglomeration State Of Silver And Titanium Dioxide Nanoparticles On Cellular Response Of Hepg2, A549 And THP-1 Cells. Toxicol Lett 2012, 208 (3), 197-213. 35. Zhu, X. M.; Wang, Y. X.; Leung, K. C.; Lee, S. F.; Zhao, F.; Wang, D. W.; Lai, J. M.; Wan, C.; Cheng, C. H.; Ahuja, A. T., Enhanced Cellular Uptake Of Aminosilane-Coated Superparamagnetic Iron Oxide Nanoparticles In Mammalian Cell Lines. Int J Nanomedicine 2012, 7, 953-964. 36. Guo, D.; Li, J.; Xie, G.; Wang, Y.; Luo, J., Elastic Properties Of Polystyrene Nanospheres Evaluated With Atomic Force Microscopy: Size Effect And Error Analysis. Langmuir 2014, 30 (24), 7206-7212. 37. Virtanen, O. L.; Mourran, A.; Pinard, P. T.; Richtering, W., Persulfate Initiated Ultra-Low CrossLinked Poly(N-Isopropylacrylamide) Microgels Possess An Unusual Inverted Cross-Linking Structure. Soft Matter 2016, 12 (17), 3919-3928. 38. Decuzzi, P.; Ferrari, M., The Role Of Specific And Non-Specific Interactions In Receptor-Mediated Endocytosis Of Nanoparticles. Biomaterials 2007, 28 (18), 2915-2922. 39. Pastan, I.; Willingham, M. C., The Pathway Of Endocytosis. In Endocytosis, Springer: 1985; Pp 144. 40. Besterman, J. M.; Low, R. B., Endocytosis: A Review Of Mechanisms And Plasma Membrane Dynamics. Biochem J 1983, 210 (1), 1. 41. Doherty, G. J.; Mcmahon, H. T., Mechanisms Of Endocytosis. Annu Rev Biochem 2009, 78, 857902. 42. Sahay, G.; Alakhova, D. Y.; Kabanov, A. V., Endocytosis Of Nanomedicines. J Control Release 2010, 145 (3), 182-195. 43. Dutta, D.; Donaldson, J. G., Search For Inhibitors Of Endocytosis: Intended Specificity And Unintended Consequences. Cell Logist 2012, 2 (4), 203-208. 44. Ivanov, A. I., Pharmacological Inhibition Of Endocytic Pathways: Is It Specific Enough To Be Useful? In Exocytosis And Endocytosis, Springer: 2008; Pp 15-33. 14 ACS Paragon Plus Environment

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45. Iversen, T.-G.; Skotland, T.; Sandvig, K., Endocytosis And Intracellular Transport Of Nanoparticles: Present Knowledge And Need For Future Studies. Nano Today 2011, 6 (2), 176-185. 46. Ivanov, A. I., Pharmacological Inhibitors Of Exocytosis And Endocytosis: Novel Bullets For Old Targets. In Exocytosis And Endocytosis, Springer: 2014; Pp 3-18. 47. Linares, J.; Matesanz, M. C.; Vila, M.; Feito, M. J.; Goncalves, G.; Vallet-Regi, M.; Marques, P. A.; Portoles, M. T., Endocytic Mechanisms Of Graphene Oxide Nanosheets In Osteoblasts, Hepatocytes And Macrophages. ACS Appl Mater Inter 2014, 6 (16), 13697-13706. 48. Hirota, K.; Terada, H., Endocytosis Of Particle Formulations By Macrophages And Its Application To Clinical Treatment. In Molecular Regulation Of Endocytosis, Ceresa, B., Ed. 2012; Pp 413-428.



Manuscript Figures and Tables Figure 1. AFM images of rhodamine-labelled pS-co-NIPAM microgels. (Scale bar = 1 µm)

Figure 2. Force-indentation plots measured with AFM nanoindentation for all hydrated pS-coNIPAM microgels at 37 oC. Solid black line is the JKR fit to the force-indentation data.


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Figure 3. Young’s moduli for all hydrated pS-co-NIPAM microgels at 37 oC determined using AFM nanoindentation.

Figure 4. MTS viability results (relative to untreated controls) for a.) RAW264.7 cells and b.) HepG2 cells treated with 150 µg/mL pS-co-NIPAM for 24 hrs.



Figure 5. a.) Dose-response curves for the uptake of each microgel in RAW264.7 cells after a 4 hr exposure, and b.) the kinetics of pS-co-NIPAM uptake (dose = 90 µg/mL) in RAW264.7 cells. (N=8 in triplicate, p

Mechanosensitive Endocytosis of High-Stiffness, Submicron Microgels

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