Cytotoxic Effects of Phosphonate-Functionalized Mesoporous Silica

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Cytotoxic Effects of PhosphonateFunctionalised Mesoporous Silica Nanoparticles Nandita Menon, and David T. Leong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11741 • Publication Date (Web): 07 Jan 2016 Downloaded from http://pubs.acs.org on January 14, 2016

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ACS Applied Materials & Interfaces

Cytotoxic Effects of Phosphonate-Functionalised Mesoporous Silica Nanoparticles Nandita Menon, David T. Leong* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore. ABSTRACT: In this work, we synthesized pristine mesoporous silica nanoparticles (MSN) and functionalised these with phosphonate groups (MSN-Phos). We report, for the first time, cell death in MCF-7 cells (human breast adenocarcinoma cell line) when exposed to the empty MSN and MSN-Phos nanoparticles. In comparison, the same nanoparticles were found to elicit few deleterious effects on normal human foreskin fibroblast cells (BJ cells). MCF-7 cells were found to exhibit a concentration-dependent uptake, while no detectable nanoparticle uptake was observed in the BJ cells, irrespective of treatment dosage. A disruption of the cell cycle in the MCF-7 cells was determined to be the cause of cell death from the nanoparticle exposure, thereby suggesting the role of non-drug loaded MSN and MSN-Phos as effective anti-cancer drugs.

KEYWORDS: mesoporous silica nanoparticles, phosphonate groups, MCF-7 cells, BJ cells, cell cycle, anti-cancer drugs.

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INTRODUCTION Breast cancer ranks among the top few diseases to affect women worldwide1. Typically, breast cancer treatment involves surgery followed by chemotherapy, hormone therapy or radiotherapy. Once administered, drugs encounter several barriers, either in the form of highly acidic tumor microenvironment that can affect the structure and/or functioning of the drug or in the form of the reticuloendothelial system (RES) that rapidly clears drugs from the bloodstream2, 3. Several important anti-cancer drugs clinically used are hydrophobic in nature and their low solubility in aqueous media makes it a herculean task to directly administer them intravenously4. Doxorubicin (DOX, trade name Adriamycin5) is highly efficacious towards cancer and despite its relatively high hydrophobicity and therefore low solubility in physiological blood, it is administered intravenously in combination with two other hydrophilic anti-breast cancer drugs cyclophosphamide (trade name Cytoxan) and 5-fluorouracil (5-FU)5 to treat breast cancer. This combination of free drugs introduced into the patient while reducing the overall necessary dose of DOX and increasing its hydrophilicity, continues to be a major cause of severe side-effects since the drug combination is administered systemically and thus does not target tumor cells specifically. To overcome this issue of drug non-specificity and thereby effectively reduce the overall dose of drugs administered to patients, drug delivery systems are designed with targeting capabilities to deliver the toxic cargo only to the tumor cells while sparing the normal cells6. Nanomaterials (materials comprising of nanoparticles that have a size range of 1-100nm79

) meet the size threshold that favors a quick cellular uptake10-13 for faster response time and

reduces extracellular release, while exhibiting augmented therapeutic efficacy courtesy the enhanced permeability and retention effect or EPR14-16. Nanomaterials are also endowed with several useful properties like quantum size effects and high surface-to-volume ratio17, 18 which


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allows for widespread application in biomedical applications, in both categories of diagnostics and therapeutics19-25. Mesoporous silica nanoparticles (MSNs) with their nano-scale particle size and high surface area to volume ratio are an ideal candidate for anti-breast cancer drug delivery to tumor cells. These features also enable MSNs to bear multiple functionalities that can be used to improve loading capacity, targetedness or for bioimaging purposes10. Several studies have focused on efficient loading of MSN and controlled, targeted delivery of DOX specifically; Zhao Q. et al. used hyaluronic acid (HA) as a capping agent and conjugated these on the MSN surface by glutathione (GSH) cleavable disulfide bonds26. Prior to HA conjugation, thiol-functionalised MSN was loaded with DOX, recording 19.8% drug loading efficiency. Hyaluronidases (HAase) provided the enzyme-responsive release of HA coating while higher concentrations of GSH in tumor cells acted as an internal redox stimulus to break the disulfide bonds. In another study, Zou Z. et al. developed DOX-loaded MSN that was first functionalised with folic acid (FA), then coated with a layer of gelatin and specked with polyethylene glycols (PEG)27. Loading amount of DOX in these MSN-FA@Gelatin-PEG nanoparticles was recorded at 74.3mmol per gram of SiO2. Tumor tissues that are reported to have upregulated MMP-2 (a type of matrix metalloproteinase, MMP) levels28 hydrolyzed the gelatin layer effectively deshielding the PEG moieties and removing the protective coating over the MSN cargo drug molecules. The FA was now made available to facilitate folate-receptor mediated uptake29 of the drug-loaded nanoparticles by the tumor cells27. Several studies have been conducted to record the effects of DOX-loaded MSN specifically on MCF-7 cells; each of these studies has either modified aspects of the MCF-7 cells to use a simplistic MSN model, or conjugated MSN with different functional groups or coating


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agents. Wang X. et al., in their study, developed MCF-7 cells that overexpressed members of the adenosine triphosphate binding cassette (ABC)-transporters (known to be associated with the multidrug resistance (MDR) phenotype in breast cancer cells). When exposed to DOX-loaded MSN over duration of 48 h, these MCF-7/MDR cells exhibited a half-maximum inhibitory concentration as low as 7.38µg/ml30. In another study, Rim H. P. et al. coated DOX-loaded MSN with pH-sensitive calcium phosphate to act as pore blockers and recorded upto 30% cell viability in MCF-7 cells when exposed to these nanocarriers over a 24 h time period31. Li L. L. et al. designed polyvalent MSN-aptamer bioconjugates that were functionalised with phosphonate groups internally to load DOX and the aptamer on the external surface was used to target MCF-7 cells. This group recorded IC50 values of 2.4 M for phosphonatefunctionalised, DOX-loaded MSN-Pho nanoparticles and 990 nM for DOX-loaded, aptamerbearing ‘MSN-Pho-Apt’ nanoparticles thereby indicating higher therapeutic efficacy when conjugating DOX-loaded MSN with cell targeting aptamers32. However, in this paper, we challenge the assumption that the vehicle (here, MSN and MSN-Phos) is passive and benign. We hypothesized that since at the molecular scale drugs behave as a web of interactions (albeit a complicated one) between functional groups and intracellular molecules, we can have a much simpler system of just one functional group (phosphonate) to bring about higher cytotoxicity towards cancer cells. The implications of any enhanced cytotoxicity suggested that perhaps we should now explore molecular scale interactions and use ever-simpler functional groups to increase cytotoxicity instead of only climbing up the slippery slope of complicated multiple-functional group systems. Here we synthesized mesoporous silica nanoparticles (MSN) and functionalised the mesopores with phosphonate groups (MSN-Phos), and tested the uptake of these nanoparticles


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and their cytotoxic effects on MCF-7 cells and BJ cells. We chose to work with BJ cells, a human fibroblast cell line, because tumorous tissues are known to be surrounded by fibroblast cells which aid in malignant progression of these tumorous cells33. We observed a concentrationdependent uptake of both MSN and MSN-Phos nanoparticles in the MCF-7 cells in 24 h that resulted in time- and concentration-dependent cytotoxic effect in these cells. In comparison, no detectable nanoparticle uptake was recorded in BJ cells and hence, there was no significant cytotoxic effect of the nanoparticle treatment on these cells. Towards the next step of this study, we loaded both nanoparticles with the hydrophobic, anti-cancer drug, DOX, and compared loading efficiencies. Our study demonstrated improved DOX loading efficiency of the nanoparticles post surface functionalisation; we recorded 28% increase in DOX loaded per unit area of nanoparticle in MSN-Phos as compared to pristine MSN. A combination of our findings projects the potential of mesoporous silica nanoparticles as simplistic anti-breast cancer drugs, and the ability to bind to an established anti-breast cancer drug, DOX, leading to higher therapeutic efficacy of the system.

EXPERIMENTAL SECTION Materials and Measurements: Cetyltrimethylammonium chloride solution (CTAC, 25wt.% of water), tetraethyl orthosilicate (TEOS, 98%), anhydrous sodium acetate (>99%), silicone oil and sodium borohydride (NaBH4) were all purchased from Sigma-Aldrich. Glacial acetic acid (100%) was purchased from Merck. Doxorubicin hydrochloride (DOX) was purchased from Apollo Scientific Limited. 3(Trihydroxysilyl)propyl methylphosphonate monosodium salt solution (THMP, 50wt.% in H2O) was purchased from Axil Scientific. Technical grade ethanol (99.86% w 5% methanol) was


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purchased from Aik Moh Paint & Chemicals Pte Ltd and analytical grade ethanol from Fischer Chemical. High-glucose Dulbecco's Modified Eagle's Medium (DMEM) containing L-glutamine supplemented with 10% FBS and 1% antibiotic/antimycotic solution was purchased from PAA Laboratories. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was purchased from SigmaAldrich. Dimethyl sulfoxide (HPLC grade, 99%) was purchased from Alfa Aesar. Nitric acid (HNO3, 65%) was purchased from Merck. Paraformaldehyde (PFA) and Triton X-100 were purchased from Sigma-Aldrich. Phalloidin (CF568 conjugate) was purchased from Biotium and Fluorescein isothiocyanate–dextran (FITC-Dextran) was purchased from Sigma-Aldrich. All chemicals were used without further purification. Phosphate-buffered saline (PBS; PAA Laboratories was used to prepare nanoparticle stock suspensions. Breast cancer cells (MCF-7) and Human neonatal foreskin fibroblast cells (BJ) were obtained from American Type Culture Collection (ATCC, Manassas, VA). All experiments in this study were done in triplicate. Data are mean ± standard deviation (SD). The statistical significance was ascertained following Student’s t-test with significant level of p < 0.05. Synthesis of Mesoporous Silica Nanoparticles MSNs within the range of 55–70 nm (Figure 1.) were synthesized using the micelle-templating method (Scheme S1.) as described previously34. The nanoparticles were functionalised with phosphonate groups using a procedure wherein after 2 h of surfactant CTAC micelle formation was allowed, 3.47ml of silica precursor TEOS was added drop-wise under constant stirring and 15 min later, 0.88ml of THMP was added to introduce the phosphonate groups (MSN-Phospristine)35,

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on the MSN pore walls. The subsequent steps were identical to the synthesis of

MSN-pristine. The nanoparticles were next calcinated to remove residual surfactant molecules


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(Figure S1.), and chemically etched (Scheme S2.) using sodium borohydride to increase pore diameters37 and denoted MSN-E and MSN-Phos-E. Transmission electron microscopy (TEM; JEOL 2010) was used to determine particle size and morphology, and dynamic light scattering (DLS; Malvern, UK) to measure hydrodynamic size and zeta potential. Cellular Uptake of Nanoparticles and Measurement of Cell Viability To observe the uptake of nanoparticles by cells, MCF-7 and BJ cells were treated with FITCtagged MSN-E and MSN-Phos-E at varying concentrations for 24 h, and prepared for analysis under a confocal laser scanning microscope. To analyze the uptake of nanoparticles quantitatively, MCF-7 and BJ cells were treated with varying concentrations of nanoparticles for 24, 48 and 72 h and analysed using the ICP-MS analysis technique. To ascertain reduced side-effects from the treatment procedure38, it was imperative to establish non-toxicity of the empty carrier nanovehicles on the normal BJ cells. After treatment of the cells with MSN-E and MSN-Phos-E, they were hence subject to the MTT Assay to measure cell viability. Gene Expression Profiling and Cell Cycle Analysis To understand the mechanism of action of the MSN on the MCF-7 and BJ cells, they were subjected to nanoparticle treatment (250µg/ml) for 48 h and 72 h, and analysed for any changes in the expression of select genes (Table S1) using the qPCR. Based on the results of this study, the cells were exposed to 250µg/ml of nanoparticles for 48 h, and studied to track their progression through the various stages of the cell cycle to understand discrepancies in this function. The Tali® image based cytometer was used to perform the cell cycle analysis.


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RESULTS AND DISCUSSION TEM images showed that calcinated nanoparticles (MSN and MSN-Phos) and etched nanoparticles (MSN-E and MSN-Phos-E) were spherical in shape (Figure 1. A-D). Hydrodynamic radius for the etched nanoparticles were measured to record the actual size of these nanoparticles when introduced to cell cultures. When dispersed in complete culture medium, DMEM, the true sizes of nanoparticles exposed to the cells were determined as shown in Figure 2. A.

Figure 1. TEM images of (A) MSN, (B) MSN-E, (C) MSN-Phos and (D) MSN-Phos-E were analyzed to determine respective primary particle sizes. Data represents mean ± SD, n=3. Scale bar = 50nm. An increase in hydrodynamic particle size, as indicated by Figure 2. A, can be attributed to particle aggregation in test dispersant system; nanoparticles have previously been reported to


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readily absorb proteins in serum-supplemented cell culture media creating a protein corona on the particle surface leading to increased diameters39, 40. The presence of Mg2+ and Ca2+ ions in cell culture media is also postulated to play a role in nanoparticle aggregation41, 42. Zeta potential is indicative of the surface charge carried by the nanoparticles; values recorded less than 30mV indicated reduced electrostatic repulsion between the particles leading to a tendency to form aggregates in DMEM43, a characteristic that can be observed in the nanoparticles synthesized, from Figure 2. B.

Figure 2. Hydrodynamic radius (A) and zeta-potential (B) of the nanoparticles before and after etching; the nanoparticles were measured in DMEM complete media. Data represents mean ± SD, n=3. High specific surface area and large pore volumes are characteristics of the mesopores on MSNs that allow accommodation of a variety of functional groups and cargo molecules44. Hence BET analysis was conducted to calculate the specific surface areas of the mesopores (BrunauerEmmett-Teller (BET) method) present on the MSNs of the nanoparticles synthesized (Table S2.), and their average pore diameters and pore volumes (by the Barrett-Joyner-Halanda (BJH)


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method, Figure S2.) to ensure the nanoparticles synthesized are physically capable of holding the phosphonate groups and DOX molecules within the mesopores. It was observed that the average pore diameter of the nanoparticles increased in the nanoparticles post chemical etching process (Table S2.). The total pore volumes of MSN and MSN-E have been recorded to be higher than those of MSN-Phos and MSN-Phos-E. A similar difference is noted in the values of specific surface area recorded for the nanoparticles wherein the MSN and MSN-E have recorded higher values in comparison to MSN-Phos and MSN-PhosE (Table S2.). The XPS analysis was conducted on MSN-E and MSN-Phos-E to determine the presence of Si2p and O1s peaks in both samples, and the presence of P2p peak only in the MSN-Phos-E sample (Figure S3.) indicating successful functionalisation of the phosphonate groups. We loaded the nanoparticles with Doxorubicin hydrochloride to observe that the etched nanoparticles achieved higher loading efficiencies than those whose pore diameters were not modified (Table S3.). However, it was also observed that the loading efficiency of phosphonate functionalised particle was approximately 49% lower than that of the non-functionalised counterpart. This can be explained by a closer examination of the surface characteristics of the nanoparticles; MSN-E exhibit a higher specific surface area than that of MSN-Phos-E (Table S2.) which directly translates to MSN-Phos-E carrying higher amount of DOX per unit surface area (Figure S4.) indicating a better interaction of the MSN-Phos-E nanoparticles with the DOX molecules. As observed from the results of the MTT assay, after treating MCF-7 cells with the nanoparticles for 48 and 72 h, a concentration- and time-dependent decrease of cell viability in these cells was recorded (Figure 3. A, C). In comparison, the BJ cells exhibited normal cell


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growth after 48 h of treatment and only a slight drop in cell viability after 72 h of nanoparticle exposure (Figure 3. B, D).

Figure 3. The figures present MTT assay results on cell viability of MCF-7 (A) and BJ cells (B) against a concentration dependent scenario of nanoparticle treatment and the same for MCF-7 and BJ cells against a time dependent scenario (C, D). Data represents mean ± SD, n=3, *p

Cytotoxic Effects of Phosphonate-Functionalized Mesoporous Silica

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