Article pubs.acs.org/molecularpharmaceutics
Shear Stress and Its Effect on the Interaction of Myoblast Cells with Nanosized Drug Delivery Vehicles Leticia Hosta-Rigau and Brigitte Stad̈ ler* iNANO Interdisciplinary Nanoscience Centre, Aarhus University, Denmark S Supporting Information *
ABSTRACT: An important aspect to ensure progress in biomedicine is the fundamental understanding of the interaction of cells and tissue with (bio)materials. The consideration of shear stress in drug delivery and/or tissue engineering remains largely unexplored. To illustrate the fundamental relevance, we employ a microfluidic setup to evaluate the myoblast cell response to two prominent drug carrier systems, namely, liposomes and nanoparticles, in the presence of low shear stress. We show that positively charged carriers have an enhanced interaction with myoblast cells in the presence of shear stress. This effect can be translated into improved therapeutic response in terms of reduction in cell viability when delivering a cytotoxic compound or into a better translocation efficiency when using lipoplexes. Taken together, our fundamental findings open up new possibilities in tissue engineering and drug delivery by considering an additional parameter when delivering beneficial compounds. KEYWORDS: drug delivery, shear stress, liposomes, cell viability, translocation
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endothelial cells than high-avidity ones, that flow has an effect on the uptake pathway, and that there is a difference between chronic and acute shear stress. A comprehensive study by Samuel et al. reported on the interaction of endothelial cells with negatively charged quantum dots and small silica nanoparticles.16 In contrast to the literature involving targeted nanoparticles, they found that low shear stress led to an increased particle uptake followed by a decrease in uptake with increasing shear stress. Furthermore, it was proposed that there is an optimal shear stress to increase the number of bound polyplexes to cells,11 and that the toxicity of 50 nm mesoporous particles was slightly increased in the presence of shear stress.10 While all these reports point toward the importance of shear stress in the context of drug delivery, to the best of our knowledge, there is no systematic assessment of the uptake/ association efficiency of typical drug delivery vehicles with other cell types than endothelial cells. The correlation with therapeutic responses in the presence of shear stress with the potential to be beneficial in tissue engineering remains largely unexplored. Here, the aim is to evaluate, from the fundamental point of view, the myoblast cell response to important drug carriers and the subsequent therapeutic response when the administration happens in the presence of low shear stress utilizing a microfluidic setup. Specifically, we (i) compared the association
INTRODUCTION Understanding the fundamental interactions of cells and biological tissue with (bio)materials is of paramount importance to ensure advances in biomedicine from tissue engineering to drug delivery. Since the circulatory system of the human body consists of kilometers of blood vessels of different diameters (arteries, capillaries, and veins) and contains ∼5 L of circulating blood under directional flow with different flow rates to provide oxygen and nutrients to the organs and cells in the body while removing “waste products”, shear stress has long been documented as an important factor in living systems. Shear stress has been shown to be involved in cellular processes such as the activation of receptors.1 In tissue engineering, shear stress has been proven beneficial for the quantity and quality of tissue obtained, in particular for blood vessel and cartilage.2−4 Recently, the relevance of shear stress has also started to be recognized in drug delivery. For instance, shear stress activated nanocarriers for local release of cargo in obstructed vessels have been reported.5,6 There are also very few prior reports which assess the basic interaction of a delivery vehicle with endothelial cells by taking shear stress into consideration.7−16 These findings are particularly important for intravenous drug administration, since drug delivery vehicles will inevitably get in contact with the dynamic environment of the circulatory system. Among these first results are increased transfection efficiency8,9 and decreased targeting of particles7,13,14 in the presence of shear stress. Han et al. identified more parameters important in cell/nanoparticle interaction in the presence of shear stress.15 They found that low-avidity antibody functionalized particles exhibit a more flow dependent interaction with © XXXX American Chemical Society
Received: March 5, 2013 Revised: May 23, 2013 Accepted: May 28, 2013
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dx.doi.org/10.1021/mp4001298 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
Polymer Labeling. PLL was fluorescently labeled by mixing FITC (∼0.3 mg in 300 μL of DMSO) and a solution of PLL in pH 10 sodium bicarbonate/NaOH buffer (∼30 mg of PLL in 6 mL). The reaction was allowed to proceed overnight, after which time the polymer was purified via gel filtration and isolated via freeze-drying. Coating of 300 nm Diameter Silica Particles. For positively charged particles (P+), a suspension of 300 nm diameter SiO2 particles (5 wt %) was washed 3 times in HEPES buffer (1200 g, 1 min) followed by sonication (1 min) after each wash. The particles were suspended in a PLL-FITC solution (5 mg mL−1, 15 min), washed 3 times, and resuspended in HEPES buffer. For negatively charged particles (P−), the previously coated silica particles were incubated with a PMA solution in HEPES buffer (5 mg mL−1, 15 min), washed 3 times, and resuspended in HEPES buffer. For both samples, the concentration of particles was determined by flow cytometry using a C6 Flow Cytometer (Accuri Cytometers Inc.) and an excitation wavelength of λ = 488 nm. At least 20 000 particles were analyzed. The particle suspension was added to the cells at a ratio 1:64 (cell/particle). Lipoplex Preparation. Lipoplexes were prepared by mixing Lipofectamine and Oligo-FITC (0.02 mM in H2O) in a ratio 1.3:1 v/v in DMEM supplemented with only 1 mM sodium pyruvate (as recommended by the manufacturer’s instructions). The mixture was incubated for 20 min at room temperature prior to addition. Cell Experiments. The C2C12 mouse myoblast cell line (ATTC) was used for all the experiments. The cells (175 000 cells/flask in 20 mL of medium) were cultured in 75 cm2 culture flasks in medium (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg mL−1 penicillin, 50 μg mL−1 streptomycin, and 1 mM sodium pyruvate, all purchased from Invitrogen) at 37 °C and 5% CO2. To perform short-term (30 min long) continuous flow experiments, cells were seeded at a density of 10 000 cells/ channel in 120 μL of medium into closed perfusion chambers (μ-slide VI0.4, tissue culture treated, Ibidi GmbH, Munich, Germany) and at a density of 7500 cells/well in 200 μL of medium onto a 96-well plate (96-wp) and allowed to attach for 24 h at 37 °C and 5% CO2. 8 mL of cell medium (with all the supplements) containing the sample (L, LTC, P, TC, or lipoplex) in the desired concentration was added to the reservoir (syringe) of the pumping system as depicted in Figure 1A. The perfusion chamber was connected to a syringe pump and put in the incubator (37 °C and 5% CO2) during the flow experiment using two different shear stresses, τ1 = 0.0146 dyn cm−2 (τ1 = 0.00146 Pa) and τ2 = 0.146 dyn cm−2 (τ2 = 0.000146 Pa), for 30 min. As controls, 120 μL of sample containing medium was added to the next channel of the perfusion chamber and to three wells of the 96-wp, and 120 μL of cell medium without sample was added to the third channel and to 3 wells of the 96wp and incubated under static conditions (τ0 = 0 dyn cm−2) for 30 min. Cell Association/Uptake Experiments. After the incubation time, for cell association experiments using liposomes, particles, or lipoplexes, the tubings were removed after the incubation time and the channels and the wells were flush-washed with PBS (100 μL) 2×, followed by the addition of 60 μL of trypsin to detach the cells from the surface for 5 min. The cells were harvested by washing the channels with PBS (100 μL) 2× and
of differently charged liposomes and nanoparticles with myoblasts, (ii) assessed the viability of these cells when a small cytotoxic compound trapped in liposomes or free in solution is delivered, and (iii) evaluated the translocation of a fluorescein labeled dsRNA oligomer using myoblast cells. The difference between the presence and absence of shear stress is a key factor considered in all the above-mentioned assessments.
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EXPERIMENTAL SECTION Materials. Sodium chloride (NaCl), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), hydrochloric acid (HCl), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), poly(L-lysine) (PLL, Mw 40 000−60 000), poly(methacrylic acid) (PMA, Mw 18 600), paraformaldehyde (PFA), and chloroform were purchased from Sigma-Aldrich. Silica particles (300 nm diameter) were obtained from Microparticles GmbH, Germany. Fluorescein isothiocyanate (FITC), BLOCK-iT Fluorescent Oligo (Oligo-FITC, 1 mM), and Lipofectamine 2000 Transfection Reagent (Lipofectamine) were purchased from Invitrogen. Zwitterionic lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, phase transition temperature −2 °C, 25 mg mL−1), negatively charged lipids 1-palmitoyl-2-oleoyl-snglycero-3-phospho-L-serine (sodium salt) (POPS, phase transition temperature 14 °C, 10 mg mL−1), positively charged lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC, 10 mg mL−1), and fluorescent lipids 1-oleoyl-2-[6[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero3-phosphocholine (NBD-PC, 1 mg mL−1) dissolved in chloroform were obtained from Avanti Polar Lipids, USA. Thiocoraline (TC) was isolated and purified by PharmaMar, S.A. (Colmenar Viejo, Madrid, Spain). Water from the Millipore water system (Milli-Q gradient A 10 system, resistance 18 MΩ cm, TOC < 4 ppb, Millipore Corporation, USA) was used for all the experiments. Liposome Preparation/Characterization. Unilamellar liposomes were prepared by evaporation of the chloroform of the lipid solutions (2.5 mg of POPC (Lzw), POPC/POEPC = 10:1 (L+,10%) or 4:1 (L+,20%), or POPC/POPS = 10:1 (L−,10%) or 4:1 (L−,20%)) under vacuum for 1 h followed by hydration with HEPES buffer (10 mM HEPES and 150 mM NaCl, pH 7.4) and extruded through 100 nm filters 11 times to obtain liposomes of monodisperse size. For fluorescently labeled liposomes, 1% (w/w) of NBD-PC was added to the lipid solution, and the fluorescence intensity of the liposome suspension at an emission wavelength of 464 nm when the sample was excited at a wavelength of 531 nm was measured using a multimode plate reader (PerkinElmer) and adjusted to the same value for the independent repeats by diluting the sample. For TC loaded liposomes, 0.1 mg of TC dissolved in chloroform was added to the lipid mixture. The final loaded amount of TC was quantified by fluorescence spectroscopy by exciting the LTC solution at a wavelength of 365 nm and recording the fluorescence intensity at an emission wavelength of 547 nm using a multimode plate reader. The concentration of TC was calculated by correlation with a calibration curve (Supporting Information Figure S4). The diameter (z-average value) and polydispersity (PDI) of the liposomes were measured using a dynamic light scattering (DLS) instrument (Zetasizer nano, Malvern Instruments) and found to be ∼150 nm and
