Tuning Endothelial Permeability with Functionalized Nanodiamonds

Tuning Endothelial Permeability with Functionalized Nanodiamonds Magdiel I. Setyawati,† Vadym N. Mochalin,‡ and David T. Leong*,† †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore ‡ Department of Chemistry, Missouri University of Science & Technology, Rolla, Missouri 65409, United States S Supporting Information *

ABSTRACT: Cancer nanomedicine vehicles are required to cross the vascular barrier to reach the tumor site in order to ensure the successful delivery of their therapeutic load. Here, nanodiamond (ND) variants were shown to induce surface dependent vascular barrier leakiness. The ND-induced leakiness was found to be mediated by the increase in intracellular reactive oxygen species (ROS) and Ca2+. These then in turn triggered the loss in endothelial cell−endothelial cell connections of the vascular barrier and also triggered their quasi-stable cytoskeletal remodelling. This ND driven increase in leakiness allowed more doxorubicin drug to penetrate through the vascular barrier to reach the cancer cells. This increase in the doxorubicin penetration subsequently led to an increase in the cancer killing effect. Overall, tuning the vascular barrier leakiness through ND surface group functionalization could provide an alternative strategy for the cancer nanomedicine to traverse across the vascular barrier. KEYWORDS: nanodiamond, endothelial cells, vascular barrier leakiness, cancer drug delivery, nanomedicine

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ancer nanomedicine encompasses drug delivery vehicles, imaging and contrast agents aimed at cancer treatment and diagnosis.1−8 Despite having all the advantages associated with the nanometer scaled carrier size (e.g., higher drug loading, longer retention in the blood circulation, pathological site targeting, and multimodality),1,9 the promise of nanomedicine has not materialized into widespread clinical practice yet. The cancer survival rate and the major cause of cancer mortality, the cancer drug delivery inefficiency, remains unchanged since the inception of cancer nanomedicine 50 years ago.1,10 The inefficiency stems from the need of the nanocarriers to cross the vasculature barrier (posed by endothelial cells) for the nanomedicinal strategy to work. While much design effort was spent on how to target, image and kill the tumor, comparatively little is dedicated to understand the upstream process of even reaching the tumor in the first place. It has been widely assumed that enhanced permeability and retention (EPR) effect could help the nanomedicine cross that barrier, as the leaky tumor vasculature allows passive and selective penetration of circulating nanomedicine to the tumor site.11 Nevertheless, strategies relying on the EPR effect resulted in a very small portion of the administered nanomedicine actually ending up at the tumor site.11 This is because the degree of leakiness of the tumor vasculature varies significantly within the same tumor site,10 © 2015 American Chemical Society

leading to heterogeneous drug delivery. In addition, the EPR effect varies according to tumor locations and even cancer progression stage.10,12 Finally, the EPR effect is tumor derived and the nanotechnologist is literally at the mercy of the tumor if one solely relies on the EPR effect for delivery of the nanoparticles to the tumor. Without this critical control over the leakiness in the tumor vasculature, any advanced nanomedicine would have little opportunity to exert its intended effect on the tumor because it could not even reach the tumor. Thus, there is a need to move away from the over-reliance on the EPR effect as the sole means of accessing the tumor. Previously, we found that titanium dioxide nanomaterials (TiO2 NM) could induce the opening of the paracellular route of the vascular barrier through their interaction with vascular endothelial cadherins (VE-cadherins), the junction proteins that regulate the paracellular route.13 This inspires us to design NM that could modulate the loosening of the paracellular route, allowing the nanotechnologist to control the leakiness in targeted vasculature and open up a novel avenue of accessing the tumor. In essence, we sought to come out with a Received: October 15, 2015 Accepted: December 7, 2015 Published: December 7, 2015 1170

DOI: 10.1021/acsnano.5b06487 ACS Nano 2016, 10, 1170−1181

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Figure 1. Physiochemical characterization of ND variants. (A) Schematic representation of ND variants surface characteristic. (B) Transmission electron microscopy (TEM) images show the irregular structure of the ND variants. Scale bar: 50 nm. (C) Hydrodynamic size distribution ND variants in ultrapure water (dashed line) and protein-rich environment (supplemented EndoGRO, continuous line). Lower panel: summary of measured ND hydrodynamic properties in ultrapure water and cell culture medium (supplemented EndoGRO). Data are means ± S.D., n = 3.

RESULTS AND DISCUSSION ND Characterization. Our hypothesis centers on tuning the surface properties (Figure 1A) of the ND variants to induce the vascular barrier leakiness that could assist drug delivery across the vascular barrier. NM physicochemical properties are known to act as the determinants in many nanobio interaction processes,14,15 which makes understanding the physicochemical properties of the ND variants, a pivotal part of the study. Transmission electron microscopy (Figure 1B) reveals that all the ND variants possess irregular shape with primary size of 7− 11 nm. This is in line with the previously reported size of detonation synthesized NDs to be ∼5 nm,18 in addition this shows that the surface functionalization did not alter the ND primary size. NM interaction with cells occurs in the cell-NM interface, where NM colloidal behavior is one of the deterministic factors of the initiation of this interaction. Hence, the ND hydrodynamic size and zeta potential (ζ-potential) within aqueous environment and in the protein-rich environment were analyzed. Our dynamic light scattering (DLS) analysis (Figure

nanotechnological strategy that would induce controllable leakiness. Much of the nanomedicine efficacy is determined by their physiochemical properties, in particular their surface properties that govern the initial interaction between the nanomedicine and the cells.14,15 As such, we hypothesized that by tuning the surface properties of our NM model we could modulate the opening of the paracellular route, resulting in the higher efficacy of cancer drug delivery. In order to prove our hypothesis, we chose nanodiamonds (ND) that are known for their tunable surface structures.16 In addition, ND is wellknown for their biocompatibility16−18 and its efficacy as carrier for various cancer therapeutic drugs,18−20 making them an excellent candidate for this strategy. Here, we show by way of changing the chemical groups on the ND’s surface, that we could modulate the degree of the paracellular route opening in the vascular barrier. This resulted in control over the amount of cancer drugs that successfully crossed over the vascular barrier and their subsequent therapeutic efficacy. In addition, the mechanisms that mediate the ND-induced permeability of the vascular barrier were elucidated. 1171


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Figure 2. ND variants induce leakiness on the vascular barrier. (A) Untreated control group showed a continuous layer of endothelial cells with no apparent gaps. Subsequent addition of ND variants showed dose dependent increase in gaps formation (red arrowheads). Adherens junction protein, VE-cadherin, was visualized with immunofluorescence (green) and the cells’ nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B) Quantification of the various degree of leakiness performed on Transwell assay showed the dose and time dependent trend with NH2-functionalized NDs group showing the greatest degree of leakiness among the three groups tested. Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, *Significant against untreated control.

found to have the largest hydrodynamic size, size, an indication of agglomeration, probably due to insufficient electrostatic repulsion that helps to maintain the colloidal stability. However, the contrast in ND’s surface characteristics that we observed in the aqueous environment vanished in the protein rich cell culture environment. The ζ-potential of all three ND variants was registered at similar value of −24 mV. This shift could be caused by the protein corona formation due to protein absorption on the ND surface.14,15 The protein corona formation was also apparent from the increase in the hydrodynamic size of the ND and ND-COOH. Interestingly, the ND-NH2 showed decrease in its hydrodynamic size, in line with the previous reports suggesting that the protein corona could act as dispersant of the NM particles, preventing the formation of NM aggregates by steric hindrance.25 ND Variants Induce Leakiness on the Vascular Barrier. We observed that the ND variants could induce intercellular gap formation on the otherwise confluent vascular barrier formed by the endothelial cells (Figure 2A). The leakiness was confirmed by growing a monolayer of endothelial cells on Transwell insert to mimic the vascular barrier in the blood vessel. We observed that the ND-induced vascular leakiness was manifested as early as 1 h following the ND variants exposure, as evidenced by the increase of fluorescein isothiocyanateconjugated dextran (FITC-dextran) that successfully crossed over the vascular barrier (Figure 2B). The FITC-dextran penetration across the vascular barrier was detected to steadily increase concomitantly with the increase of ND variants’ dose and time of exposure (Figure 2B). As intact vascular barrier only allows minimal FITC-dextran penetration, this increase of

1C) shows that the ND hydrodynamic diameter is bigger than their primary size due to aggregation of the ND in the aqueous environment (ultrapure water, pH 6.8). NM colloidal stability is heavily influenced by its surface charge and NM with ζpotential less than ±30 mV tends to form aggregates in aqueous solution.21,22 The aggregation of ND variants was well supported by our ζ-potential readouts which were registered approximately at −16, −26.6, and −10.1 mV for the ND, NDCOOH, and ND-NH2, respectively. The ND negative ζpotential is indicative of the presence of oxygen containing functional groups on ND surface (mainly COOH, CO, OH).16 As the ND processed with air oxidation to obtain the ND-COOH, the ζ-potential value swung further to the negative region, confirming the conversion of the partially oxidized groups into the carboxylic groups on the ND surface.18,23,24 Further attachment of ethylenediamine (EDA) via the amide bond (FTIR characterization and ζ-potential values were previously reported24) onto the ND-COOH surface resulted in the formation ND-NH2 that could be detected by the notable shift of ζ-potential to become less negative. The shift was caused by the protonation of the NH2 groups in aqueous environment. It is worth noting that the process to produce ND-NH2 did not completely convert all carboxyl groups into the amide groups (the amino group coverage approximation is 20% of the ND-NH2 total surface).24 As such, the ζ-potential value of ND-NH2 was still within the negative value range and not totally shifted toward the positive region, as one might expect if the ND surface is terminated completely with NH2 groups. In agreement with our hydrodynamic size measurement, ND-NH2 with the smallest value of ζ-potential value was 1172


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Figure 3. ND-induced vascular barrier leakiness is modulated by the intracellular reactive oxygen species (ROS). (A) All three ND variants induced a dose- and time- dependent increased ROS level. The highest production of oxidative species was triggered by ND-NH2 exposure. (B) The ND-NH2 (3h exposure) induced the highest percentage of cells producing mitochondrial (Mt) superoxide. Antioxidants, N-acetyl cysteine (NAC, 1 mM) and glutathione (GSH, 10 mM) (C,D) alleviated the production of oxidative species. (E) ND variants triggered ROSmediated production of the intracellular Ca2+. (F) Antioxidant attenuated the vascular barrier leakiness. This identifies the ROS control over the ND-induced leakiness. Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, *Significant against untreated control, #Significant against no antioxidant group.

NM. Hence, it is possible that the difference in the leakiness degree that we observed was caused due to the different amount of ND variants available to interact with the vascular barrier to begin with. Differences in NM surface profile and aggregation size are attributed to the different electrostatic and steric repulsion forces that influence the degree of NM colloidal stability in the aqueous environment.26,27 This in turn leads to different amount of NM available near the cell surface to interact with the cell. Nevertheless, our ND deposition study (Figure S1) disputed the possibility that the difference in leakiness degree was caused by the different amount of ND available on the cell surface. We observed that ND variants during the leakiness time frame (1, 3, and 6 h) had a similar deposition profile (Figure S1) with a small margin of difference between the deposited ND variants. We detected maximum difference between the deposited ND variants to be 4%, which occurs at the longest exposure time (6 h). Considering the small size of the ND primary particles, one could argue that with 4% margin, the difference would amount to sufficient ND particles at the cell surface to engage the cells. If the amount of

FITC-dextran penetration can only suggest the increased vascular barrier permittivity due to the gaps formed. Though all ND variants were found to induce vascular barrier leakiness, we consistently detected that ND-NH2 induced the highest degree of leakiness (3.5-fold). In contrast, the vascular barrier exposed to ND-COOH shows the least leakiness (2.4-fold), as can be observed in Figure 2B. As the ND variants used here were produced from the same well characterized initial material23 only differing in their surface modification and taking into account that their colloidal behavior in cell culture medium was similar, any difference in the observed leakiness must be attributed to the distinct differences in surface chemistries of these ND variants. This finding reinforces our initial hypothesis that vascular barrier leakiness could be induced by the ND itself and its surface functionalization. More importantly, this also convinces us that through a rational design of ND surfaces, we could obtain the desired control over vascular barrier leakiness. As previously mentioned, the NM induced biological response is initiated at the interface between the cell and the 1173


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Figure 4. ND variants trigger activation of ROS-modulated intracellular pathways that results with the ND-induced vascular barrier leakiness. (A) NDs variants activated Akt pathway and its subsequent downstream event of (B) nitric oxide production. (C) ND variants induce phosphorylation of VE-cadherin (P-VEC), signaling for its internalization and degradation. (D) The addition of Src kinase inhibitors, PP1 (10 μM), helps to attenuate leakiness. Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, *Significant against untreated control, #Significant against no PP1 treated group. (E) ND variants activated of ERK pathway. The involvement of ERK pathway in the ND-induced leakiness was evident from the significant reduction in the vascular barrier leakiness upon addition of ERK inhibitor, PD98059 (10 μM). Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, *Significant against untreated control, #Significant against no PD98059 treated group. (F) ND variants reduced the acetylation in tubulin, signing the microtubule remodeling. (G) Actin remodeling was required in the ND-induced vascular barrier leakiness. Blocking RhoA kinase with Y-27632 (10 μM), significantly reduced the ND-induced leakiness. Green and blue fluorescent signals represent the VE-cadherin and DAPI stain, respectively. Red arrowheads mark the gaps formation on the vascular barrier. Scale bar: 50 μm. Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, *Significant against untreated control, #Significant against no Y-27632 treated group.

of ND-based nanomedicine, the mechanistic knowledge could identify, reduce and even prevent potential nanomedicine side effects on the patients and this safer-by-design paradigm in turn increases its chance for clinical approval. In order to obtain the holistic view of the ND-induced leakiness, we turned our focus to the reactive oxygen species (ROS) and the calcium ion (Ca2+) due to their role as main regulators to many cellular responses and in particular the regulation of barrier leakiness.28−30 Furthermore, studies have shown that NM could initiate the cross-talk between the intracellular ROS and Ca2+.31−33 It also has been noted that the differences in the NM surface characteristics may influence the level of intracellular ROS production.31,34 Thus, we asked whether the different degree of vascular leakiness could be linked to the difference in ROS production profile as the causal effect of ND distinct surface characteristic. To address this question, we studied the level of intracellular ROS production. We observed that the

the deposited ND variants were the deciding factor of the leakiness, we could expect the highest degree of leakiness to be observed in the ND-COOH treated group that has the highest percentage of particle deposition (22.4% within 6 h; Figure S1). The data, however suggested otherwise. Though being deposited in the highest amount, ND-COOH group induced the least leakiness on the vascular barrier (Figure 2). It seems that the vascular barrier leakiness was not derived solely from the ND-cell interaction that occurs at the cell surface but possibly required the activation of intracellular processing. ROS Mediated Intracellular Pathways Control the NDInduced Vascular Barrier Leakiness. Though we have shown the efficacy of ND in inducing leakiness on vascular barrier, there is a significant knowledge gap in the induction mechanism. A clear understanding of how the ND-induced leakiness came about could guide nanobiotechnologist to further tune its efficacy. In addition to pushing the development 1174


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connection to the eventual cellular response (i.e., the barrier leakiness) is not yet established. Taking into account the identified ROS/Ca2+ involvement, we turned to the downstream process within these modulators’ purview that could help explain the mechanism. The vascular barrier is held together by a set of homophilically interacting adherens junction proteins, VE-cadherins. These homophilically interacting VE-cadherins that join one endothelial cell to its neighbors are buttressed by the cytoskeleton networks.30,36 In order for the leakiness of the vascular barrier to occur, the VE-cadherin homophilic interactions have to be interrupted and the support provided by the cytoskeleton has to vanish.30 It has been noted that vascular endothelial growth factor (VEGF) and thrombin utilize intracellular Ca2+ through protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) signaling pathways to signal for junction protein disassembly.30,37−39 To examine the involvement of Akt and eNOS signaling, we probed the cellular transduction following the endothelial cells exposure to the ND variants. Exposure to ND variants resulted in the phosphorylation of Akt protein at the position of Serine 473, indicating the activation of the pathway (Figure 4A). We also noted the significant dose dependent elevation of intracellular nitric oxide (NO) production (Figure 4B, Figure S3) following the increase in dose of ND variants. Consistently, we observed the much higher induction of NO production by the cells exposed to ND-NH2 (5.9-fold) when compared to the ND and ND-COOH (4.3- and 3-fold, respectively). As eNOS is capable to catalyze the NO production in the endothelial cells, the increase of NO production indicates the ability of ND to activate the eNOS signaling. It has been reported that in response to increased influx of Ca2+, endothelial cells activate Akt pathway that in turn signals the eNOS to ramp up the NO production.38 Furthermore, we detected that the ND variants triggered phosphorylation of VE-cadherin at its sites of tyrosine 658 and 731 (Figure 4C). In response to the phosphorylation, the VEcadherin is retracted and degraded intracellularly, leaving no available VE-cadherins to form the homophilic interactions required to maintain the integrity of vascular barrier.30,36 To conclusively prove that the Akt/eNOS/VE-cadherin signal transduction is involved, we inhibited the activity of Src kinase, which is responsible for the phosphorylation and that in turn activated the Akt/eNOS/VE-cadherin signal transduction.40 Pretreatment of endothelial cells with PP1 (10 μM), the Src kinase inhibitor, successfully brought the level of protein phosphorylation to its basal level, as evidenced from the VEcadherin phosphorylation level (Figure S4). Concomitant to the phosphorylation inhibition, the permeability index was significantly reduced (Figure 4D). This clearly showed that eNOS induced by NDs brought about vascular barrier leakiness through the activation of the signaling transduction axis of Akt/ eNOS/VE-cadherin. However, the loss of adherens junction interconnections from the extracellular portion of the endothelial cells is not enough to induce increase in the vascular barrier permeability. Though sufficiently blocking the Akt/eNOS/VE-cadherin signaling resulted in a significant decrease of the ND-induced vascular barrier permeability, it was not fully abolished (Figure 4D). This indicates that Akt/eNOS/VE-cadherin signaling only partially explained the mechanism of ND induced barrier permeability. As mentioned previously, the vascular barrier leakiness also requires the loss of cytoskeleton support. Control over the cellular cytoskeleton network is notably known to be

intracellular ROS production in the endothelial cells (Figure 3A) increased concomitantly to leakiness (Figure 2). Consistent with our previous observation, the ND-NH2 induced the highest ROS production, as evidenced by the 4fold increase of ROS production when compared to ND and ND-COOH that only induced 2.7- to 3-fold increase of ROS production, respectively (Figure 3A). The ROS elevated level could be contributed to two sources: (1) extracellular, in which the ND is highly reactive and able to produce ROS independent of the cells and (2) intracellular, in which the cells produce ROS as a response to the ND exposure. In a cell free environment, we did not observe any ROS generation detected for all ND variants (Figure S2), suggesting that the cells’ intracellular processing was responsible for the elevated ROS level we observed. In order to further ascertain this, we checked for mitochondrial activity. Mitochondria are recognized as the primary site for intracellular ROS production,35 due to their capability to generate superoxide species. This species goes through a dismutation reaction, resulting in hydrogen peroxide (H2O2), the major component of ROS, as the end product.35 If the elevated level of ROS that we detected is produced intracellularly, then we would expect to observe a corresponding increased activity of the mitochondria. Indeed, we observed a significant shift in the population of mitochondrial superoxide producing cells (Figure 3B). Semiquantitatively, we detected 12- to 22- fold increases in the superoxide production after exposure to NDs in 400 μg/mL concentrations, compared to the untreated control group. This result further supports that the elevated ROS level was truly produced intracellularly as a biological response toward the ND variants. Furthermore, it was noted that the ND-NH2 variant triggered the most number of cells to produce the mitochondrial superoxide, which means upon dismutation reaction, we could expect a larger increase in the H2O2 production that in turn contributed to the increase of intracellular ROS as observed in Figure 3A. Antioxidants, Nacetyl cysteine (NAC, 1 mM) and glutathione (GSH, 10 mM) were found to reduce the production of intracellular oxidants, bringing their level close to the basal level (Figure 3C, D). ROS has been established to control the barrier permeability through intracellular Ca2+.30,33 In our intracellular Ca2+ assessment, we found significant increase in intracellular Ca2+ after the cells were exposed to the ND variants (Figure 3E). In order to establish the crosstalk between the ROS and Ca2+ level and the contribution of this cross-talk to controlling the NDinduced leakiness of the vascular barrier, we once again employed the antioxidants, NAC and GSH, and performed rescue experiments. We observed that the level of intracellular Ca2+ returned to its basal level (Figure 3E), establishing that ROS and Ca2+ level are indeed interconnected. More importantly, concomitant with the reduction of intracellular ROS and Ca2+ production, we observed that the ND-induced vascular barrier leakiness was significantly attenuated with the addition of the antioxidants (Figure 3F). As these antioxidants work by scavenging the intracellular ROS, it is expected that the introduction of these antioxidants would considerably bring down the oxidant species production, maintain the Ca2+ homeostasis, and finally attenuate the ND-induced vascular barrier leakiness. This provides strong evidence that ROS level adjustments (by way of Ca2+) were involved in the ND-induced vascular barrier leakiness. Though we have clearly identified ND-induced ROS production as the main modulator for the leakiness, its 1175


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Figure 5. ND-induced vascular barrier leakiness assists cancer therapy by promoting penetration of cancer drug across the vascular barrier. Top panel: Experimental scheme. (1) Vascular barrier was treated with ND variants to induce leakiness. (2) Following the induction of leakiness the ND variants were removed and the leaky vascular barrier was transferred to another well in which the MDA-MB-468 cancer cells were grown and (3) the doxorubicin (DOX) was added. (4) The excess DOX was removed along with the EC, followed by quantification of the amount of DOX successfully penetrating the vascular barrier and the DOX effect on the MDA-MD-468. Bottom panel: (A) ND variant treatments promotes the penetration of DOX across the vascular barrier. (B) Increase cancer cell (MDA-MB-468) cytotoxicity concomitant with the increase of DOX penetration over leaky vascular barrier. Fresh cell culture medium and DOX (320 nM) were added to single culture ̂ MDA-MB-468 cells to serve as negative and positive controls, respectively. Data are means ± S.D., n = 3, Student’s t-test, p < 0.05, Significant against untreated vascular barrier. *Significant against untreated MDA-MB-468 group.

contraction.44 If the actin rearrangement were a vital part of the ND-induced leakiness, one would expect reduced barrier leakiness after blocking this rearrangement. Incubation of the endothelial cells with Y-27632, as expected, reduced the formation of stress fibers which provided intracellular contractile force without disturbing the barrier integrity (Figure S6). Both immunofluorescence and transwell data (Figure 4G) showed the reduction of ND-induced vascular barrier leakiness when the cells’ capability to remodel their actin filaments was curtailed. This indicated that the actin remodeling process is an integral part of the ND-induced vascular barrier leakiness. It is worth noting that blocking either the Akt/eNOS/VE-cadherin or ERK signal transduction only resulted in a partial recovery of the leaky vascular barrier (Figure 4D,E). This further supports our initial hypothesis that the ND-induced leakiness cannot be explained by only the loss of adherens junction interconnection, but also involves remodeling of the cytoskeleton network. ND-Induced Vascular Barrier Leakiness Assists Cancer Therapy. To demonstrate the efficacy of our strategy to induce leakiness of the vascular barrier in assisting the delivery of cancer therapy, we conducted a proof of concept study. Here, we formed a vascular barrier and treated it with ND variants (200 μg/mL, 3 h) to obtain the leaky vasculature barrier model prior to exposing it to anticancer drug doxorubicin (DOX) acting on breast cancer cells MDA-MB-468. We detected only a small amount of DOX diffused through the untreated vascular barrier, whereas significant increase of DOX was detected to traverse the vascular barrier that was pretreated with the ND

held by extracellular signaling regulated kinases (ERK) signaling pathway, which is known to be mediated by ROS.30,40,41 ND variants exposure resulted in activation of ERK pathway, as evidenced by the significant phosphorylation of the ERK (Figure 4E). Inhibiting the ERK pathway with its specific inhibitor, PD98059 (10 μM), successfully suppressed the ERK phosphorylation (Figure S5) and significantly attenuated ND-induced vascular barrier leakiness (Figure 4E). This supported the involvement of the ERK pathway to transduce the signal to bring about ND-induced leakiness. Further downstream of ERK, we detected the remodeling of two important cytoskeletal networks, microtubules (Figure 4F) and actin filaments (Figure 4G) in the endothelial cells exposed to the ND variants. The microtubule remodeling could be detected by the reduction of the acetylated tubulin expression level after cell exposure to ND variants (Figure 4G). The deacetylation of the microtubule, which is catalyzed by the ERK-modulated histone deacetylase, indicates the microtubule network destabilization, its disassembly and reorganization.42 Another cytoskeletal network that has been implicated with endothelial barrier permeability is actin filament. The actin cytoskeletal remodeling is controlled by RhoA kinase (ROCK) whose activation is triggered by the ERK pathway.41,43 To investigate the involvement of the actin cytoskeleton remodeling in the ND-induced leakiness, we perturbed the ROCK signaling pathway with the cell permeant Y-27632 (10 μM). Y-27632 suppresses ROCK capability to control actin cytoskeleton rearrangement that is required for the cell 1176


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Figure 6. Schematic illustration of ND-induced vascular barrier leakiness. ND-induced vascular barrier leakiness allows more doxorubicin to reach the tumor site. The ND-induced leakiness was modulated by the increase of intracellular ROS and Ca2+. This leads to the loss of cell− cell interconnection on the vascular barrier and the cytoskeletal remodeling, two requisite events for vascular barrier leakiness.

this deliberate induction of vasculature leakiness could be achieved to different degree by NDs with distinct surface profiles. The leakiness induction was observed to be highly affected by the ND variants’ surface profile with ND-NH2 showing the highest degree of leakiness induction. Central to the ND-induced leakiness is the intracellular ROS, whose production was contingent to the NDs variants’ surface profile and their internalization. We have shown the effect of this distinct ROS production level on the downstream signaling that could lead to the ND-induced leakiness. Yet, one question that still begs to be answered is how the different surface groups on these ND variants could affect the ROS production level. One possibility is the intracellular ROS difference could be directly linked to the surface groups found on the ND variant itself. ND is invariably decorated with partially oxidized surface groups which may get oxidized inside the cells and release oxidants as a byproduct of the reaction. In contrast, the fully oxidized ND-COOH does not have the means to release oxidative species, leaving the net ROS production of ND to be higher than the ND-COOH. Notable intracellular oxidation process upon NM have been reported for silver (Ag) NM, in which upon their internalization, the Ag NM get further oxidized to Ag+ ions, producing significant amount of ROS byproducts.31,46 ND-NH2 with prominent NH2 group on its surface could engage the intracellular amine oxidase that utilizes amine groups as substrate and releasing ROS as one of its conversion products.47 It is also plausible that surface groups on the ND variants indirectly affect the extent of their internalization, resulting in the different ROS production levels in the cells. Though it may seem to be in contradiction with our observation of ND deposition, it is possible that the different surface groups decorating the ND attract different types of proteins in the process of the protein corona formation. Depending on the nature of the protein, the interaction between the ND-protein complex and the cells could be promoted or hindered, resulting in the difference in the internalization route and the amount of particles being internalized.14,48,49 For example, protein corona surrounding positively charged polystyrene (PS) nanoparticles facilitated its

variants (Figure 5A). In a parallel experiment, we detected incremental increase in cell death (ca. 6%) for the MDA-MB468 cells that were exposed to the DOX over untreated vascular barrier when we compared to the untreated MDA-MB-468 (Figure 5B). We noted improvement in the killing effect of DOX when the vascular barrier was pretreated with the ND variants. The killing effect improvement was registered to be approximately 87%, 60%, and 140% following pretreatment of vascular barrier with ND, ND-COOH, and ND-NH2 , respectively (Figure 5B). This trend again demonstrates the higher potency of ND-NH2 to induce vascular leakiness compared to two other ND variants. We did not detect any perceivable vascular leakiness when the endothelial cells monolayer and the MDA-MB-468 cells were cocultured (Figure S7), confirming that the increase in cell death is attributable to the ND-induced vascular barrier leakiness. This is in line with our previous transwell data (Figure 2B) showing that following ND variants’ treatment, the vascular barrier became leaky and allowed a more unrestricted solute transport across it, which in this case resulted in more DOX crossing the barrier per unit of time and exerting its killing effect on the cancer cells. Highest DOX migration over the vascular barrier that brought about highest killing effect was noted for MDA-MB-468 cells that had ND-NH2 treated endothelial cells as their vascular barrier (Figure 5). This supports our overall observation that the surface properties of ND variants indeed play the dominant role in the vascular leakiness induction. Passive targeting of therapeutics via the EPR effect in cancer is solely dependent on the tumor and never on the nanomaterial. The network of hastily formed and immature tumor vasculature is unpredictable. The leaky tumor vasculature only manifests after aberrant angiogenesis, which occurs at the mid to late stage of tumor progression.10,12,45 In contrast, in the earlier stages where the tumor is more responsive toward any drug treatment, the vasculature around this “easier to kill” tumor is unfortunately not leaky. As early detection and treatment of tumors will result in a more optimistic prognosis, accessing the earlier stage tumor via artificially creating a leaky vasculature may be a viable option. We have demonstrated that 1177


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transient in nature. Our current data suggest that up to 1 h after removal of the ND variants, there was still significant leakiness to allow the migration of the DOX through the leaky barrier to the cancer cells. We did not have the real time scale required to achieve full recovery on the ND-induced vascular leakiness. Nevertheless, we suspect it would be a prolonged process, estimated from the time (8 h to more than 24 h) required to achieve full recovery on the NM induced leakiness of vascular barrier53 and gut barrier.55 Going forward, it is imperative that we could achieve recovery within the shortest time span possible, as future research is aimed toward this ultimate goal. Besides improvements from the nanomedicine side, the cell systems could be “upgraded” to 3D cell systems to give a more realistic representation of the actual in vivo biological systems.57

internalization through the scavenger receptor. In contrast, protein-PS complex derived from anionic negatively charged PS scarcely get internalized as the complex had to compete with serum protein to engage the native protein receptors.48 As ROS plays a central role in the vascular barrier leakiness, we could contemplate the use of other substances, such as few of metallic ions carefully titrated at certain concentrations, to induce the intracellular ROS production that lead to a similar effect as what we observed with ND variants, albeit through a different mechanism altogether.50 ND-induced leakiness is promoted by the increased ROS production followed by activation of its downstream pathways that lead to loss of VE-cadherin interconnection and cytoskeleton remodeling (Figure 6). The same manner of molecular transduction is also observed to be the hallmark of VEGF-induced permeability.51 As VEGF induction is initiated upon its internalization through VEGF receptor (VEGFR),36 it could be logically deduced that the ND variants act as artificial antagonist for VEGFR and utilize its interaction to the receptor to initiate the signal transduction. In recent findings, the opening of the intercellular gaps on the blood brain barrier was successfully achieved with nanoantagonist against adenosine receptor (A2R) which was designed to treat brain ischemia.52,53 In addition vascular barrier leakiness could also be promoted through the NM interaction with junction proteins that hold the barrier together. TiO2 NM induced leakiness was found to be triggered by its association with the adherens junction protein, VE-cadherin.13 In intestinal barrier setting, chitosan NM interaction with the tight junction protein, claudin-4, has been reported to induce the loosening of intercellular gaps, allowing more insulin to permeate through the loosened gaps and enter the blood circulation.54,55 The ultimate goal of understanding the induction mechanism as well as identifying the initial interaction is to allow us to finetune the leakiness induction to assist future development of ND mediated drug delivery. Our data suggest that the ND-induced leakiness requires its internalization. One possible route for the ND to enter the cells is through the caveolin mediated pathway, as this internalization route is predominantly used by the endothelial cells to take in solutes, including nanomedicine particles, from the blood circulation.14 Moreover, the VEGFlike signal transduction suggests that the VEGF receptor is engaged in the internalization process. The binding of ND and the VEGF receptor would likely trigger the cells to form the “coated pits” lined by clathrin molecules to take in the ND via the clathrin-mediated pathway.14 Clathrin-mediated pathway has been reported to actively transport the epirubicin-adsorbed ND into chemoresistant hepatic cancer stem cells.19 In addition, blocking the lipid raft formation of caveolin-mediated pathway by methyl-β-cyclodextrin significantly reduced the ND internalization into the human umbilical endothelial cells.56 Both caveolin- and clathrin- mediated pathways in many ways depend on how strong the initial binding between the ND and the receptor is. It could be surmised that by providing stronger binding, for example through VEGFR specific antibody (to target the clathrin-mediated pathway) conjugation or through albumin coating (to target the caveolin-mediated pathway), we could achieve a more pronounced leakiness induction. We envisioned that having stronger binding and a more pronounced leakiness induction could help to reduce the dose and time required for leakiness induction, minimizing possible systemic toxicity. Undoubtedly, to be safely used as nanomedicine, this ND induction of leakiness should be

CONCLUSIONS In this study, ND variants have been demonstrated to be able to induce the desired endothelial leakiness, which is found to be surface dependent. The ND-induced leakiness is mediated by the ROS. Increased doxorubicin drug penetration to the cancer cells, which in turn leads to an increased cancer killing efficacy, is found to be concomitant with the vascular leakiness induction by ND. Overall, we have demonstrated the possibility to achieve better cancer drug delivery by deliberately inducing leakiness on the vascular barrier using the diamond nanoparticles. MATERIALS AND METHODS Cell Culture. Primary human dermal microvascular endothelial cells of neonatal origin were purchased from Life Technologies, USA. Breast cancer cells, MDA-MB-468 were obtained from ATCC, USA. EndoGRO-MV-VEGF (Merck Millipore, USA) supplemented with Gentamicin (50 μg/mL; Sigma-Aldrich, USA) and Amphotericin-B (50 ng/mL; Sigma-Aldrich, USA), hereby denoted as supplemented EndoGRO, were used to culture the endothelial cells. Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin cocktail, hereby denoted as supplemented DMEM, was used to grow the MDA-MB-468. The cells were grown in standard culture condition (37 °C, 5% CO2, 95% relative humidity) and were subcultured when they reach 90% confluence. Throughout the study, the endothelial cells and MDAMB-468 cells were seeded at seeding density of 40 000 and 50 000 cells/cm2, respectively. Nanodiamond (ND) Surface Functionalization and Characterization. The untreated nanodiamond (ND) was obtained from Zhongchuan Heyuan, China. To study the role of NDs surface properties in inducing the vascular barrier leakiness, the as received ND was purified and functionalized as previously reported.18,23,24 Briefly, the nanodiamonds with carboxylic terminated groups (NDCOOH) were obtained by purifying the ND via air oxidation at 425− 430 °C for 2 h.23 The aminated nanodiamonds (ND-NH2) were produced by covalently linking ethylenediamine to the previously described ND-COOH.18,24 The morphology of ND variants was observed with field emission transmission electron microscope (FE-TEM; JEOL JEM-2100F, Japan). Briefly, the ND variants (10 μg/mL) were dispersed in analytical ethanol by way of sonication for 1 min (Qsonica micronson XL2000, USA). The ND suspensions then were dropped on the carbon coated copper grids and dried at ambient room temperature. The image of particles of ND variants was taken with the FE-TEM at the accelerating voltage of 200 kV. The primary particle size was derived from the FE-TEM micrograph, in which 50 randomly selected particles were measured for their sizes with ImageJ software.58 In addition, the ND variants were characterized using the dynamic light scattering (DLS; Malvern, UK). Briefly, the ND particles (10 μg/ mL) were dispersed in ultrapure water and the supplemented 1178


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In a parallel set of experiments, the wells were filled with supplemented DMEM and the amount of doxorubicin that successfully penetrated through the ECs barrier was measured. Following the doxorubicin exposure time (1 h), the insert containing the ECs and the excess doxorubicin was discarded and the supernatant at lower chamber was sampled. The doxorubicin content was determined by measuring the intensity of fluorescence emitted by the drug (Excitation: 480 nm, Emission: 580 nm). Statistical Analysis. All experiments were conducted in triplicate. Data are means ± standard deviations. Statistical significance was ascertained with paired sample Student’s t-test comparison. Results were considered as statistically significant with p < 0.05.

EndoGRO medium with sonication in ice bath for 1 min. The ND variants were suspended in supplemented EndoGRO medium then were spun down (21 000g, 10 min), their pellets were collected and redispersed in the ultrapure water with sonication in ice bath for 1 min. Thereafter, the suspensions were measured for their hydrodynamic size, polydispersity index, and ζ-potential. The measurement was conducted in triplicate and the average value was reported. Preparation of ND Suspension and the Cell Exposure. The ND concentrations as high as 500 μg/mL have been previously reported to be biocompatible.59 As such, the dose range of 50, 100, 200, and 400 μg/mL was chosen to be used to examine these ND variants’ capability to induce vascular barrier leakiness. The ND suspension for endothelial cells exposure was prepared by dispersion in the supplemented EndoGRO medium. The dispersion was aided by sonication in the ice bath for 1 min. The endothelial cells exposure was done by removing the growth medium and replacing it with medium containing ND variants. Fresh growth medium was used to treat the control group. For study using antioxidant or inhibitor, the endothelial cells were first pretreated with the antioxidant or inhibitor for 1 h. Following that, the pretreatment medium was removed and fully replaced with ND variants (200 μg/mL) which were dispersed in the medium containing the said inhibitor or antioxidant. Following the treatment (3 h), the subsequent assays were conducted as described in the following section. Fresh growth medium supplemented with the antioxidant or inhibitor was added in the control group. The antioxidants and inhibitors were purchased from Sigma-Aldrich (USA). The full list of the antioxidants and inhibitors which were used in the study is shown in Table 1.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b06487. Additional experimental details and data. (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by Singapore Ministry of Education Academic Research grant (R-279-000-418-112 to D. T. Leong). M. I. Setyawati was partially supported by the World Future Foundation (WFF) PhD Prize in Environmental and Sustainability Research.

Table 1 compound

final conc.

function

N-acetyl cysteine (NAC) L-glutathione reduced (GSH) PP1

1 mM 10 mM

antioxidant antioxidant

10 μM

PD98059 Y-27632

10 μM 10 μM

Src-family protein tyrosine kinase inhibitor MAPK/ERK kinase inhibitor Rho associated kinase inhibitor

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Permeability Transwell Insert Assay. The degree of vascular barrier leakiness was measured in Transwell insert (with polycarbonate filter, 0.4 μm pore; Corning Costar, USA). Briefly, the monolayer endothelial cells were cultured on the insert and exposed to ND variants for 1, 3, and 6 h. Fresh cell culture medium was used as control. FITC-dextran (1 mg/mL) was supplemented in all treatment solution/suspension. Following the treatment, 150 μL of samples were taken from the lower compartment and the fluorescence signal was quantified with a microplate reader (Biotek H4, USA) at excitation/ emission wavelength of 490/520 nm. The degree of endothelial monolayer leakiness is expressed as permeability index where the fluorescent signal from the treated group was normalized against the one collected from the control group. ND Variants Efficacy As Drug Delivery Adjuvant. Monolayer endothelial cells barrier was treated with ND variants (200 μg/mL, 3 h). At the end of exposure time, the ND variants were removed from the transwell insert, the insert then was moved to an adjacent well where MDA-MB-468 was grown in supplemented DMEM. Following that, doxorubicin (5 μM in supplemented DMEM; Sigma-Aldrich, USA) was introduced on the apical side of the insert and further incubated for 1 h. At the end of doxorubicin exposure, the insert containing the ECs monolayer and the excess doxorubicin, was discarded and the MDA-MB-468 cells were further incubated for 72 h. Following that, the cells were collected and stained with Tali Cell Death Green kit (Life Technologies, USA) and the cell viability was assessed with Tali Image based cytometer (Life Technologies, USA). 1179


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