Nanoparticle Density: A Critical Biophysical Regulator of Endothelial Permeability Chor Yong Tay,*,†,‡ Magdiel Inggrid Setyawati,§ and David Tai Leong*,§ †
School of Materials Science and Engineering, Nanyang Technological University, N4.1, 50 Nanyang Avenue, Singapore 639798, Singapore ‡ School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore § Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore S Supporting Information *
ABSTRACT: The integrity of the vasculature system is intrinsically sensitive to a short list of biophysical cues spanning from nano to micro scales. We have earlier found that certain nanomaterials could induce endothelial leakiness (nanoparticle induced endothelial leakiness, nanoEL). In this study, we report that the density of the nanomaterial, a basic intrinsic material property not implicated in many nanoparticle-mediated biological effects, predominantly dictates the nanoEL effect. We demonstrated that the impinging force exerted by a library of increasing effective densities but consistently sized silica nanoparticles (SiNPs) could directly increase endothelial permeability. The crossover effective particle density that induced nanoEL was determined to be between 1.57 g/cm3 to 1.72 g/cm3. It was also found that a cumulative gravitational-mediated force of around 1.8 nN/μm along the boundaries of the vascular endothelial cadherin (VE-cad) adherens junctions appeared to be a critical threshold force required to perturb endothelial cell−cell adhesion. The net result is the “snapping” of the mechanically pretensed VE-cad (Nanosnap), leading to the formation of micron-sized gaps that would dramatically increase endothelial leakiness. KEYWORDS: nanoparticles, nanobio interactions, particokinetics, endothelial cells, VE-cadherin mechanics, biophysics such as fluid shear stress, 8 Ca 2+ concentration, 9 and inflammatory conditions10,11 across different time and length scales. Previously, we have shown that engineered nanoparticles instead of only interacting with the cell membrane, also binds to the extracellular domain of the VE-cad that is critically necessary to connect neighboring endothelial cells.5,12 We found that inorganic nanoparticles (NPs) (TiO2, SiO2, and Ag) (primary sizes 15 to 25 nm) randomly entered into the nanometers wide gaps of the adherens junctions between endothelial cells and disrupted those important VE-cad−VEcad interactions; producing micron sized gaps between the endothelial cells.5 This “nanomaterials induced endothelial leakiness” (nanoEL) was not observed when bigger TiO2 submicron particles (668 nm) were unable to induce nanoEL, suggesting that this process is indeed specific to materials that are in the nanoscale range. Apart from the size-dependent characteristic, very little is known about the other NPs physical
E
ndothelial cells (ECs) are simple squamous cells joined at their cell borders to form the inner walls of all blood and lymphatic vessels of the human vasculature. An intact ECs layer is essential to regulate numerous fundamental functions of the endothelial barrier, such as the control of blood flow, controlled passage of materials, blood pressure, and clotting events.1 The integrity of the endothelium is highly dependent on the formation cell−cell adhesions that is mediated via a group of specialized intercellular junction complexes. Unlike the epithelial junctions, the molecular make up of endothelial cell junctions is much more complicated and is composed of tight, adherens, and gap junctions.2 A major component of the endothelial cell−cell adherens junctions is the vascular endothelial-cadherin (VE-cad), also known as cadherin 5 and CD144. VE-cad is known to play a central role in vascular development and maintenance of the restrictive endothelial barrier.3,4 A systemic loss of VE-cad not only disrupts communication between adjacent cells but also contributes to various pathophysiological conditions like metastasis and bacterial sepsis.5−7 To fulfill its role as the “gatekeeper” of the endothelium, the VE-cad is able to respond dynamically to changes in the perivascular microenvironment, © 2017 American Chemical Society
Received: November 20, 2016 Accepted: March 13, 2017 Published: March 13, 2017 2764
DOI: 10.1021/acsnano.6b07806 ACS Nano 2017, 11, 2764−2772
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Figure 1. Physical characterization of silica nanoparticles (SiNP) with varying effective density. (A) TEM pictograph of the pristine mesoporous silica nanoparticles (MSN) and the associated (B) N2 adsorption−desorption isotherm. From which the internal pore volume (Vint) can be determined at P/P0 = 0.9. The mesopores were then backfilled with predetermined amount of TEOS to attain a panel of SiNPs (C to F) with different amount of Vint and therefore effective density, ρE. Scale bar = 50 nm.
Table 1. Physiochemical Characteristics of the Synthesized SiNPsa average size (nm)
a
sample
TEOS added (μM/mg of SiNP-0)
SiNP-0 SiNP-1 SiNP-2 SiNP-3 SiNP-4
2.02 4.03 6.05 8.06
TEM 48.1 50.9 52.8 58.3 61.6
± ± ± ± ±
5.6 4.3 4.8 5.6 7.3
ζ (mV)
DLS 63.5 77.8 77.9 87.6 88.6
± ± ± ± ±
4.3 2.5 3.4 9.2 3.8
−18.6 −25.1 −23.5 −20.2 −17.7
± ± ± ± ±
0.5 0.6 0.5 1.3 0.5
Vint (cm3/g)
ρE (g/cm3)
0.236 0.181 0.126 0.071 0.016
1.45 1.57 1.72 1.90 2.13
TEM: Transmission Emission Microscopy; DLS: Dynamic Light Scattering; Vint: Internal Pore Volume; ζ: Zeta Potential.
and nanoparticle size that could influence nanoEL should be kept ideally constant. The principle of varying particle density begins with mesoporous SiNPs (SiNP-0) as the “ground state” or base material. To increase the density of the SiNP, we “backfilled” the mesopores with different amount of silica precursor under stringent synthesis conditions. As can be seen in the TEM pictograph of SiNP-0 (Figure 1A), the assynthesized SiNPs are generally well-dispersed with a uniform primary particle size of approximately 48 nm. Presence of irregularly patterned mesopores (∼2−3 nm) were clearly evident within the spherical SiNPs.13 Figure 1B depicts the nitrogen adsorption−desorption isotherms of the SiNP-0 which shows a type IV isotherm with H1-type hysteresis loop which is typical for mesoporous adsorbents. SiNPs with increasing ρE (Figure 1C−F) showed a gradual decrease in mesopores size and number (from Figure 1C−F) with a slight increase (
