Mesoporous Silica Nanoparticles as an Antitumoral-Angiogenesis

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Mesoporous Silica Nanoparticles as an Anti-Tumoral-Angiogenesis Strategy Magdiel Inggrid Setyawati, and David Tai Leong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12524 • Publication Date (Web): 02 Feb 2017 Downloaded from http://pubs.acs.org on February 3, 2017

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ACS Applied Materials & Interfaces

Mesoporous Silica Nanoparticles as an AntiTumoral-Angiogenesis Strategy Magdiel I. Setyawati†*, David T. Leong†* †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore

KEYWORDS Anti-angiogenesis, mesoporous silica nanoparticles, nanomedicine, cancer, migration, invasion,

ABSTRACT Tumors depend heavily on angiogenesis for nutrient derivation and their subsequent metastasis. Targeting tumor induced angiogenesis per se can address both tumor growth and progression simultaneously. Here, we show that we could elegantly restrict the endothelial cells angiogenic behavior through digital size control of mesoporous silica nanoparticle (MSN). This anti-angiogenesis effect was derived from the particle size dependent uptake and production of intracellular reactive oxygen species (ROS) that directly interfered with p53 tumor suppressor pathway. The resulting signaling cascade wrestled back the tumoral control of endothelial cells’ migration, invasion and proliferation. Overall, a mere control over the size of a highly oxidative reactive surfaced nanoparticle could provide an alternative strategy to curb the tumor induced angiogenesis process in a conventional drugfree manner.

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INTRODUCTION Despite intensive research over the past decades, cancer still accounts for more than 8 million deaths every year.1 Majority of this cancer mortality is attributable to cancer metastasis.2 For metastasis to even begin, it has to be supported by blood vessels close to the cancer site.2-4 Cancer cells hijacked these blood vessels and induced sprouting of new capillary-like blood vessels as an escape route for metastatic cells to leave the primary tumor site and metastasize to another organ.3-4 Inhibiting this cancer induced angiogenesis process should logically slow down metastasis. Current nanomedicine strategies in angiogenesis management focus on inhibiting the angiogenesis signaling through the delivery of angiostatic compounds to the cancer cells.5-7 These strategies may well be promising anticancer strategies but does little to the prevent angiogenesis of endothelial cells. Moreover, cancer cells have been reported to circumvent certain anti-angiogenesis therapy by either releasing other type of angiogenic compounds to activate the targeted pathway or activating a different angiogenic signaling cascades altogether.7-8 In reality, only a very small percent of surviving pre-metastatic cells will be enough to eventually kill the host. So we turn the problem around and proposed to address the cancer angiogenesis issue not from the cancer cells but from the endothelial cells perspective which tend to be more amiable to therapeutic drugs. Moreover, the scope of problem does not necessitate killing any endothelial cells; resulting in a milder condition and translating to fewer side effects to the patient. Controlling nanomedicine physicochemical properties is one of the important tenets in designing nanomedicine formulation. This level of control confers advantageous characteristics to nanomedicine to allow them to be loaded with therapeutics (e.g., drugs, photodynamic therapy agents, and imaging agents),9-12 modified with targeting moieties13-14


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and encapsulated with stealth rendering compounds to ensure their prolonged circulation.15-16 In addition, tailored physicochemical properties could elicit certain response from the cell.16 By tailoring the chemical groups on nanodiamonds, we could remotely control the ROS levels of endothelial cells to cause vascular leakiness without killing the endothelial cells.17 In this report, we again exploited intracellular ROS to control endothelial cells’ proliferation and migration processes, with the intent of controlling endothelial cells’ angiogenic response to cancer cells. We propose to achieve this by tuning surface area of mesoporous silica nanoparticles (MSN) for ROS production and catering their sizes to improve cell uptake. With an optimal surface area to size combination, we expect to induce optimum cellular ROS level to cue the inhibition of endothelial cells angiogenesis. Here, we demonstrated in a powerful manner by simply changing MSN size, we could modulate both important functional traits of endothelial cells’ proliferation, migration and survival. Moreover, we elucidated the mechanism that governs the MSN induced anti-angiogenesis. Overall, this work forms the nanomaterialistic basis for more sophisticated designs or more ambitious outcomes of bionanotechnology through a better integrated approach to understand nanobiological mechanism first.

RESULTS AND DISCUSSIONS MSN characterization TEM micrographs revealed that the synthesized silica nanoparticles were spherical and porous in nature, confirming the formation of desired MSN (Figure S1A). In addition, the nanoparticles mesoporous nature was evident from the characteristic type IV isotherm hysteresis loop on the obtained nitrogen adsorption-desorption profile (Figure S1B).

18

MSN

had the average primary sizes of 41 nm (MSN40), 60 nm (MSN60), and 103 nm (MSN100) 3 ACS Paragon Plus Environment


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(Figure S1A, Table 1). The size difference between MSN groups was also observable by their specific surface area and total pore volume. Both the specific surface area and specific total pore volume was reduced as the MSN size increased (Table 1). The size increase within the MSN groups was also observed with dynamic light scattering (DLS) analysis (Table 1). MSN probably had aggregated in aqueous environment (ultrapure water, pH 6.8). This could be expected as the ζ-potential for these MSN was registered approximately at -18 mV (Table 1), which was below the requisite ζ-potential (±30 mV) to maintain the optimum colloidal stability.19-20 This negative ζ-potential could be attributed to the hydroxyl groups on their surface.21 Similar ζ-potential values were observed when the MSN groups were introduced in the protein rich environment of cell culture medium, averaging at -17 mV. Though the values were only slightly different from those registered in ultrapure water environment, these negatively charged values were predominately contributed by the presence of the protein corona formed on the MSN surface16,

22

. The detectable outcome of this protein corona

formation was the decrease in the hydrodynamic size of the MSN groups (Table 1) in the medium that rich with proteins. This could be expected as protein corona on the MSN surface could sterically hinder the MSN particles from aggregating, markedly lowering the overall hydrodynamic size.23-24

Table 1. Physical characterization of MSN.

MSN40

MSN60

MSN100

Primary size (nm)

41.4 ± 7.64

60.4 ± 8.31

103.2 ± 10.4

SBET (m2/g)

376.32

183.72

106.53

Vp (cm3/g)

0.62

0.42

0.34

in water

130.1 ± 1.9

201.2 ± 21.56

231.4 ± 50.38

in medium

99 ± 25.46

131.8 ± 6.43

141.1 ± 12.59

-17.4 ± 0.36

-18.5 ± 0.1

-20.9 ± 0.12

Hydrodynamic size (nm)

ζ-potential (mV) in water


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in medium

-20.5 ± 0.15

-14 ± 1.27

-16.8 ± 0.51

SBET: Brunauer–Emmett–Teller (BET) derived surface area; Vp: total pore volume.

As the MSN synthesis was facilitated by cationic surfactant, cetyltrimethylammonium chloride (CTAC), it is imperative to remove the residual CTAC prior to investigating the MSN efficacy as anti-angiogenesis agent. Through FTIR analysis (Figure S1C), we detected CTAC characteristic C-H stretching at 2920 and 2850 cm-1 on as-synthesized MSN samples. Nevertheless, these stretching peaks were absence on the acid-extracted MSN, suggesting the complete removal of residual CTAC. In addition, we conducted a control experiment in which we collected the supernatant from the ultracentrifugation of 400 µg/mL of MSN suspension in culture medium and introduced the said supernatant to the HMMEC. We argue that if the CTAC were not completely removed, the cells would be affected by its presence. However, we observed no perceptible change in the HMMEC viability following 24 h exposure to these supernatant concoctions (Figure S1D). This further supports the complete removal of CTAC from the MSN system.

MSN induced anti-angiogenesis in a size dependent manner During the tumor angiogenesis process, the endothelial cells sprout out from the existing blood vessel, spread and form new blood vessels towards the direction of tumor site.2-4 As such to prove our hypothesis, we grew human mammary microvascular endothelial cells (HMMEC) on matrigel matrix to form the blood vessel tube (Figure 1A).25 This gives us an opportunity to test out the MSN ability to inhibit endothelial angiogenesis process in a model that closely mimics the real blood vessel. We observed the human mammary microvascular endothelial cells (HMMEC), exposed to MSN for 12 h, failed to form the



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typical tube blood vessel network that was evident in the control group. Instead, the phase contrast microscopic images showed stunted blood vessel growth in the MSN treated groups. Quantitatively, significant decrease in endothelial tube length was observed in MSN-treated groups. Moreover, the blood vessel tubes formed by the MSN treated HMMEC was less branched compared to those formed within the untreated control group. This is evident from the significant reduction in the degree of branching and the tube network area coverage detected on the MSN treated groups (Figure 1B). More importantly, the MSN antiangiogenesis effect was observed to be dependent of the MSN size. MSN60 showed the highest anti-angiogenesis performance, as evident from the ~3.8-fold in the reduction of tube length when compared to the control (Figure 1B). In comparison, the MSN40 and MSN100 only registered close to 2.7-fold and 3.1-fold reductions, respectively. Similar trend was also observed on the degree of branching of the blood vessels and the network area coverage (Figure 1B). These observations suggest that MSN are able to impede blood vessel tubes formation in a size dependent manner.


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Figure 1. MSN inhibited tube formation of human mammary microvascular endothelial cells (HMMEC) in a size dependent manner. (A) Schematic of the tube inhibition assay. HMMEC mixed with and without MSN were seeded onto matrigel matrix and further incubated for 12 h prior to assessing the MSN inhibition effect on the tube formation. (B) Phase contrast images and semi-quantitative analysis show that MSN inhibited HMMEC tube formation in a size dependent manner. Scale bar: 100 µm. Data are means ± S.D., n = 3, Student’s t-test, p

Mesoporous Silica Nanoparticles as an Antitumoral-Angiogenesis

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