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Nanoparticle Density: A Critical Biophysical Regulator of Endothelial Permeability Chor Yong Tay, Magdiel Inggrid Setyawati, and David Tai Leong ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b07806 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 13, 2017
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Nanoparticle Density: A Critical Biophysical Regulator of Endothelial Permeability Chor Yong Tay,†‡* Magdiel Inggrid Setyawati,§ 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
KEYWORDS: nanoparticles, nano-bio interactions, particokinetics, endothelial cells, VEcadherin mechanics, biophysics
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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 nanoparticles could induce endothelial leakiness (nanoparticle induced endothelial leakiness, NanoEL). In this study, we report that the intrinsic 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 cross-over 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 cellcell adhesion. The net result is the “snapping” of the mechanically pre-tensed VE-cad (Nanosnap), leading to the formation of micron-sized gaps that would dramatically increase endothelial leakiness.
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Endothelial 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 are 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 disrupt communication between adjacent cells but also contributes to various pathophysiological conditions like metastasis and bacterial sepsis.5-7 To fulfil its role as the “gatekeeper” of the endothelium, the VE-cad is able to respond dynamically to changes in the perivascular microenvironment, such as fluid shear stress,8 Ca2+ 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 neighbouring 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-VE-cad interactions; producing micron sized gaps between the endothelial cells.5 This “nanomaterials induced endothelial leakiness” (NanoEL) was not observed when bigger TiO2 sub-micron
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particles (668nm) was unable to induce NanoEL, suggesting that this process is indeed specific to materials that are in the nano-scale range. Apart from the size-dependent characteristic, very little is known about the other NPs physical parameters’ influences on NanoEL. Without a better understanding of NanoEL, it is then hard for nanotechnologists to avoid the pathological effects of NanoEL or harness the exploitative effects of NanoEL in nanomedicine. We observed that there are proteins in blood that would be of similar size range as any NanoEL inducing nanoparticles and yet permeability do not spontaneously occur. We then hypothesized that it is the density of the NanoEL inducing nanoparticles that may have exerted an impinging force, strong enough to disrupt the VE-cad-VE-cad interaction. To validate our hypothesis, we have designed and constructed a library of silica nanoparticles (SiNP) with varying intrinsic effective densities (ρE) that would exert differing force magnitude on VE-cad-VE-cad at the point of impact. Understanding the physical aspects of NanoEL can allow us to control nanoparticles crossing of important biological barriers which directly determines nanotoxicity or future nanomedicine strategies. RESULTS AND DISCUSSION Intrinsic nanoparticle effective density: Critical determinant of NanoEL. While nanomaterials have many important physical interactions with biological systems like endocytosis, it was assumed that the intrinsic materialistic property of nanomaterials like density are not a consideration since these particles are almost infinitesimally light under gravity. Therefore, to deterministically define the role of this impinging force on VE-cad biology (as the result of changing ρE), other nanomaterial intrinsic parameters like surface charge 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
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material. To increase the density of the SiNP, we “back-filled” 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 as-synthesized 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 1 C-F) showed a gradual decrease in mesopores size and number (from Figure 1C-F) with a slight increase (< 10%) in the overall primary particle size of the SiNPs. For this study, five types of SiNPs with increasing intrinsic effective densities were abbreviated as SiNP-0 to SiNP-4. The measured and computed physical properties of the SiNPs are compiled and shown in Table 1. For a more realistic correlation of the SiNP properties to the biological observations, the hydrodynamic diameter (DH) and surface charge (zeta potential) of the SiNPs were further characterized in the cell culture medium. In the cell media, the SiNP had DH ranging from 63-89 nm and are all negatively charged. Compared to the primary particle size that was determined using the TEM images, the corresponding DH is consistently bigger which could be attributed to the formation of a hydration shell as well as the formation of protein corona, cover the surface of the nanoparticles.14, 15 Due to the presence of the mesopores in SiNP-0, the effective density (ρE) of the particles is thus expected to be lower than that of its solid non-porous counterpart. The total internal pore volume (Vint) of SiNP-0 was determined to be 0.236 cm3/g, based on the nitrogen adsorption-desorption isotherms at the relative P/P0 = 0.9, which corresponded to the onset of the capillary condensation in the mesopores. Accordingly, the computed ρE of SiNP-0,
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SiNP-1, SiNP-2, SiNP-3 and SiNP-4 are 1.45, 1.57, 1.72, 1.90 and 2.13 g/cm3 respectively (Table 1).
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/Po = 0.9. The mesopores were then backfilled with pre-determined amount of TEOS to attain a panel of SiNPs (C to F) with different amount of Vint and therefore effective density, ρE. Scale bar = 50nm.
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Table 1. Physiochemical Characteristics of the Synthesized SiNPs
Sample TEOS added (µM/mg of SiNP-0)
Average Size (nm) TEM
DLS
ζ (mV)
Vint (cm3/g)
ρE (g/cm3)
SiNP-0
-
48.1 ± 5.6
63.5 ± 4.3
-18.6 ± 0.5
0.236
1.45
SiNP-1
2.02
50.9 ± 4.3
77.8 ± 2.5
-25.1 ± 0.6
0.181
1.57
SiNP-2
4.03
52.8 ± 4.8
77.9 ± 3.4
-23.5 ± 0.5
0.126
1.72
SiNP-3
6.05
58.3 ± 5.6
87.6 ± 9.2
-20.2 ± 1.3
0.071
1.90
SiNP-4
8.06
61.6 ± 7.3
88.6 ± 3.8
-17.7 ± 0.5
0.016
2.13
TEM: Transmission Emission Microscopy; DLS: Dynamic Light Scattering; Vint: internal pore volume; ζ: zeta potential.
Using this library of nanoparticles with varying densities, we examined the ability of the nanoparticles to induce NanoEL as a function of its ρE. Endothelial cell leakiness is determined by using a modified transwell assay. After 30 min of nanoparticles treatment, the transendothelial FITC-dextran flux was measured as a proxy for endothelial permeability or leakiness. As shown in figure 2A, NanoEL was observed to be dependent on both the ρE as well as SiNPs dose. Specifically, we noted that significant endothelial leakiness was detected for samples that were treated with denser SiNPs such as SiNP-2, SiNP-3 and SiNP4, but not for SiNP-0 and SiNP-1. This suggest that a critical ρE is needed for the SiNPs to induce NanoEL. Beyond the threshold ρE, it was noted that endothelial permeability is dependent on the SiNPs dose, with SiNP-3 and SiNP-4 both displaying a maximum of 2-fold increase in endothelial leakiness compared to the untreated control. Consistent with the leakiness assay, we were also able to observe formation of inter-cellular micron-sized (10-30 µm) gaps within 30 min of SiNPs (~2.0 x 1011 particles/ml) treatment in groups treated with SiNP-2, SiNP-3 and SiNP-4 but not for cells
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that are treated with SiNP-0 and SiNP-1 (Figure 2B). Therefore, our results suggest that ρE is a vital physical determinant to the NanoEL process.
Figure 2. SiNPs with high ρE triggers endothelial cell leakiness. (A) Endothelial leakiness under various treatment conditions as measured using the FITC-dextran based tracer assay. NanoEL induction was observed to be dependent on treatment dose for the higher density SiNPs (SiNP-2, SiNP-3 and SiNP-4) (B) Representative high resolution fluorescence images of human microvascular endothelial cells (HMVECs) treated with 2.0 x 1011 particles/ml of SiNPs (2.5 x 1011 particles/cm2) for 30 min at 37oC. Cells were counterstained for nucleus (blue), VE-cad (green) and F-actin (red). Detection of significant endothelial leakiness and intercellular gaps were limited to SiNPs with higher ρE (i.e. SiNP-2, SiNP-3 and SiNP-4) but not SiNP-0 and SiNP-1. Untreated samples served as negative control. Scale bar = 25 µm. * denotes statistical difference at p
