Multifunctional curcumin-loaded mesoporous silica nanoparticles for cancer chemoprevention and therapy

Microporous and Mesoporous Materials 291 (2020) 109540

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Multifunctional curcumin-loaded mesoporous silica nanoparticles for cancer chemoprevention and therapy

T

Nihal S. Elbialya,b,*, Samia Faisal Aboushoushaha, Balsam Fahad Sofia,c, Abdulwahab Noorwalid a

Medical Physics Program, Physics Department, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia Biophysics Department, Faculty of Science, Cairo University, 12613, Giza, Egypt c Medical Physics, Department of Physics, Collage of Applied Science, Umm Al-Qura University, Makkah, Saudi Arabia d Medical School, King Abdulaziz University (KAU), Jeddah, Saudi Arabia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Cancer Curcumin-loaded mesoporous silica nanoparticles Drug delivery system Cancer chemoprevention

This study aims to synthesize smart mesoporous silica nanocarrier for curcumin (Cur) as a nutraceutical anticancer agent. Multifunctional PEG-MSNPs-Cur were synthesized to enhance curcumin bioavailability for the purpose of preventing and treating cancer. It can also serve as auto-fluorescence probe for molecular imaging (image guided therapy). The prepared nanocarrier has been characterized using transmission electron microscope (TEM), Fourier transform infrared spectroscopy (FTIR), Dynamic light scattering (DLS) and zeta potential. In vitro, the antitumor activity of PEG-MSNPs-Cur and Cur was investigated on two human cancer cell lines: liver cancer (HepG2) and cervical cancer (HeLa). Compared with free-Cur, PEG-MSNPs-Cur showed higher cellular uptake and significant cytotoxicity against HepG2 and HeLa cells. Furthermore, PEG-MSNPs-Cur-treated HepG2 cells exhibited marked cell cycle arrest at G2/M compared to free-Cur-treated cells. In vivo, cancer chemoprevention and therapeutic efficacy of PEG-MSNPs-Cur as well as Cur, have been evaluated using two treatment protocols: Tumor Chemoprevention Protocol (TCP) and Tumor Reduction Protocol (TRP). The results demonstrated that TCP exhibited high therapeutic efficacy over TRP. The toxicity of PEG-MSNPs-Cur were assessed by serum biochemical analysis and histopathological examination for certain vital body organs to address the biosafety issue of PEG-MSNPs-Cur. The integrated results indicated that smart multifunctional MSNPs greatly enhanced curcumin bioavailability. PEG-MSNPs-Cur offered pH-triggered drug release in an acidic pH (tumor microenvironment). Additionally, PEG-MSNPs-Cur is utilized as self-fluorescence probe that allow their tracing in the cells without the need of dyes. PEG-MSNPs-Cur could be effectively used as a safe chemopreventive and therapeutic agent.

1. Introduction Cancer is a significant healthcare problem, which is characterized by an abnormal and uncontrolled increase in cell proliferation. It is the most serious life-threatening disease worldwide [1]. According to the World Health Organization (WHO), it is expected that cancer death rates will increase to 12 million cases by 2030. Whereas, the rate of cancer diagnoses is expected to reach 24 million globally by 2035 [2]. The most conventional strategy used for cancer therapy is chemotherapy. Most cancer patients receive chemotherapy either alone or in combination with other oncological modalities. However, this approach has several limitations including the non-specific distribution of drugs in the body thus effecting both cancerous and normal cells, resulting in dose related side effects combined with low drug

*

concentration in the tumor site [3]. As a consequence, the use of natural products, that have anticancer properties such as induction of apoptosis and inhibition of tumor growth without harming normal cells, is pressing. A natural substance that is a food or food ingredient and offers a health or medical benefit is known as a nutraceutical [4]. One of the promising nutraceutical ingredients is curcumin, it is a hydrophobic polyphenol which is derived from the turmeric spice (curcuma longa plant). Turmeric spice is a popular south Asian spice that belongs to the ginger family and its active ingredient is curcumin [5]. Through the past decades, curcumin has attracted the attention of the scientific community due to its high therapeutic potential towards cancer. Curcumin has been found to inhibit carcinogenesis [6], cause disturbances in mitosis thus leading to cell cycle arrest [7], inhibit tumor initiation and progression (invasion

Corresponding author. Medical Physics Program, Physics Department, Faculty of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia E-mail addresses: [email protected], [email protected] (N.S. Elbialy).

https://doi.org/10.1016/j.micromeso.2019.06.002 Received 23 February 2019; Received in revised form 29 May 2019; Accepted 3 June 2019 Available online 08 June 2019 1387-1811/ © 2019 Elsevier Inc. All rights reserved.

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purchased from Across Organics (Morris Plains, NJ). Anhydrous dimethylsulfoxide (DMSO), penicillin streptomycin, antibiotic-antimycodic, 2.5% Trypsin (10X), Dulbecco's Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) GAMMA irradiated, all were purchased from Gibco (USA). WST–1 Cell Proliferation Reagent was purchased from ABCAM (UK). BioMid Diagnostic kits for (Creatinine (Cr), Uric Acid (UA), alanine aminotransferase (ALT), & aspartate aminotransferase (AST)) were purchased from BioMed Diagnostic (Singapore).

and metastasis) and constrain the formation of free radicals [8], as well as induce apoptosis and inhibit tumor growth in various cancer cells [9]. Additionally, curcumin acts as a cancer chemopreventive agent as it possess the potential to suppress the nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB-activation) [10]. Unfortunately, there are major obstacles that limit the usage of curcumin in cancer therapy including its low bioavailability and solubility, rapid degradation in physiological pH, inadequate tissue absorption, and rapid systemic elimination [11]. Nanotechnology has been applied to overcome these obstacles. A proposed novel solution is to provide a nanodelivery system represented by different types of nanoparticles such as: polymeric nanoparticles [12–14], solid lipid nanoparticles [12,14], metallic nanoparticles [14], and inorganic nanoparticles [15–18]. Among inorganic-based nanoparticles is mesoporous silica which displays very interesting features including a high surface area and a specific pore volume, thermal and chemical stability, as well as the capability of surface functionalization [19–22]. Mesoporous silica nanoparticles (MSNPs) internalize readily to eukaryotic cells through endocytosis with high biodegradability [23,24]. MSNPs also exhibit high biocompatibility and biosafety which can be customized to produce a nano-platform for cancer treatment management [18]. Moreover, MSNPs can deliver hydrophobic curcumin and protect it from enzymatic degradation [25]. In the last few years, attention has been drawn towards MSNPs not merely as a drug delivery system but also as a multifunctional one. Many studies have designed smart nanohybrid silica materials that exhibit great capabilities for the purpose of both drug delivery and bioimaging [26,27]. Among silica hybrid nanocarriers are polymer coated mesoporous silica which are strongly involved in the nanomedical field [28]. Xie et al., 2013, investigated the effectiveness of MSNPs for imaging and treatment of breast cancer [29]. Zhang et al., 2013, fabricated multifunctional MSNPs that offer stimuli responsive drug release as well as image guided cancer therapy [30]. C. Chen et al., 2018, constructed multifunctional MSNPs loaded curcumin which was specifically used as a targeted and controlled release drug delivery system. The developed MSNPs improve curcumin stability and biocompatibility achieving significant enhancement in their cytotoxicity against MCF-7 [31]. X. Xu et al., 2018, fabricated curcumin-polymer coated silica nanoparticles for cancer imaging and therapy. The smart MSNPs integrate many functions including a high drug loading ratio, controlled drug release and a self-fluorescent agent [32]. In this context, we will combine all the beneficial features of MSNPs together with the natural anticancer drug curcumin to develop a polymeric coated curcumin-mesoporous silica nanocarrier for the purpose of cancer diagnosis, therapy and chemoprevention. The current study aims to enhance curcumin bioavailability using smart multifunctional MSNPs. PEG-MSNPs-Cur is expected to offer pHtriggered drug release in an acidic pH (tumor microenvironment). Additionally, PEG-MSNPs-Cur is considered as a self-fluorescence probe that allow their tracing in the cells without the need of dyes. PEGMSNPs-Cur will also be used as chemopreventive and therapeutic agent as well.

2.2. Synthesis of PEGylated mesoporous silica nanoparticles loadedcurcumin (PEG-MSNPs-Cur) 2.2.1. Preparation of mesoporous nanoparticles The MSNPs were a gift from the Biophysics Department, Faculty of Science, Cairo University. Cetyltrimethylammonium bromide 0.5 g (porogen) was dissolved in 70 ml of distilled water and sonicated for complete dissolution. Then, 0.5 ml of ammonia hydroxide (28%) and 30 ml of 2-ethoxyethanol (co-solvent) were added to the porogen. The mixture was then transferred to a closed bottle and vigorously stirred, at room temperature, for 30 min. Then, 2.5 ml of tetraethyl orthosilicate (TEOS) was added to the mixture dropwise and stirred vigorously for 24 h. The mixture was centrifuged at 5000 rpm for 15 min, the supernatant was discarded and the white participate was collected. The pellet was then washed several times with distilled water. The supernatant was dispersed and stirred for 3 h in a mixture of 60 ml ethanol containing 120 μl of concentrated hydrochloric acid HCl to remove the porogen (CTAB) at 30 °C. This process (surfactant extraction) was repeated twice for complete removal of CTAB. Finally, the particles were washed twice with distilled water to remove the surfactant and then dried for 1 h at 250 °C to acquire CTAB free MSNPs [33]. 2.2.2. Preparation of curcumin loaded MSNPs (MSNPs-Cur) In order to load curcumin on to MSNPs, 8 mg of MSNPs was dissolved in 10 ml distilled water. Then, 2 mg of curcumin was added to 2 ml ethanol. The two solutions were sonicated for complete dissolution. The sonicated solution was shaken (100 rpm and 37 °C for 24 h using a water bathed shaker (Fisher Scientific, Shaker, USA). Then the sample was centrifuged (5000 rpm at 5 °C) for 30 min to discard unloaded curcumin. The supernatant was gathered for encapsulation efficiency calculations. Further washing was conducted twice by adding fresh distilled water to the pellet and centrifuged (5000 rpm at 5 °C) for 5 min. Then, the supernatant was collected and added to the previous collected one (for encapsulation efficiency calculations). 2.2.3. PEGylation of MSNPs-Cur In order to prepare PEGylated MSNPs, 24 mg of polyethylene glycol (PEG) with a molecular weight 4000 g/mol (PEG4000) was weighed and added to the previously prepared solution of MSNPs-Cur (1/1, w/v). The mixture was sonicated for complete dissolution and then stirred using a magnetic stirrer for 24 h to complete the PEG coating and collect the final formulation (PEG-MSNPs-Cur). 2.3. Characterization

2. Materials and methods

The morphology of the prepared MSNPs was imaged using transmission electron microscopy (TEM) at 200 kV (FEI, HR-TEM, Netherland) [34]. Fourier transform infrared (FTIR) spectroscopy was used to confirm the successful synthesis of MSNPs, PEG, MSNPs-Cur, and PEG-MSNPs-Cur (PerKinElmer, FTIR, US) [35,36]. The frequency range of FTIR spectral scanning was between 4000 and 400 cm−1. Dynamic light scattering (DLS) [15,37] and zeta potential (Zeta) [38] techniques were performed to measure the particle size distribution and surface charge, respectively, for the prepared samples using a zeta sizer (NICOMPTM 380 ZLS, Santa Barbara, California, USA). The samples were dissolved in deionized water and the measurements were repeated

2.1. Materials Curcumin (Cur) (≥94% curcuminoids content, ≥ 80% curcumin), Dulbecco's phosphate buffered saline, absolute ethanol, and diethyl ether ≥ 99 % were purchased from Sigma-Aldrich (USA). Ribonuclease (RNAse), Propidium Iodide Solution (PI), and Hoechst 33342 were purchased from Sigma (USA). Sodium tripoly phosphate, 0.1 N sodium hydroxide solution (NaOH), cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS, 98%), and acetic acid (AA, 98.7%) were 2

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2.6.2. Cellular uptake and intracellular accumulation For both HepG2 and HeLa cell lines, the cellular uptake and intracellular accumulation of PEG-MSNPs-Cur were carried out using fluorescence microscopy and compared with free-Cur. HepG2 and HeLa cells were cultured in two separate sets of 6-well plates at a total volume of 2 ml and incubated at 37 °C for 24 h. The attached cells were then treated individually with 72 μg/ml PEG-MSNPs-Cur and an equivalent dose of Cur. After incubation and at specified time intervals (0.5, 6, 24, 48 h), the plates were examined under a fluorescence microscope at 420 nm wavelength (LEICA, Fluorescence microscopy, Germany) [15].

three times and the standard deviation (SD) was calculated using origin 8.0 software. 2.4. Curcumin loading efficiency The optical density of different concentrations of the curcumin solution have been measured at 426 nm using UV–VIS spectrophotometer (Thermo Fisher Scientific, Genesys UV-VIS Spectrophotometer, China). To calculate the amount of curcumin encapsulated in the pores of MSNPs, the prepared PEG-MSNPs-Cur solution was centrifuged (13000 rpm at 4 °C) for 30 min (Sigma, cold centrifuge, Germany). The pellets were washed twice with 10% aqueous ethanol. The supernatant was carefully collected and its optical density was measured at 426 nm using UV–VIS spectrophotometer. The concentration of free-Cur in the supernatant was then calculated from the previously performed calibration curve. Then, after PEG coating, the amount of leakage curcumin was quantified by further sample centrifugation and the collected supernatant was spectrophotometrically measured once again. Finally, curcumin loading efficiency was calculated using the following formula [39]:

2.6.3. Cytotoxicity of free-Cur and PEG-MSNPs-Cur The antitumor activity of both Cur and PEG-MSNPs-Cur against HepG2 and HeLa cells was assessed using a WST-1 assay and an inverted light microscope (S1) [40]. 2.6.4. Cell cycle arrest HepG2 cells were cultured into three separate 6-well plates for 24 h, then the first plate was treated with 72 μg/ml PEG-MSNPs-Cur and the second plate was treated with an equivalent dose of free-Cur. While, the third plate was kept untreated (control) for 24 h. After that, the cells were centrifuged with cold PBS for washing. The pelleted cells were fixed and vortexed gently with 70% ethanol at 4 °C overnight (Fisher Scientific, VORTEX MIXER, U.S.A). The pellet was washed twice with PBS, centrifuged again at 2000 rpm for 5 min, and incubated with 50 μl of RNAse (Sigma, RNAse) for 15 min at 37 °C. Propidium Iodide Solution (Sigma P4170, PI) of 200 μl was added for the staining of the fixed cells [41]. After that, the cells were washed in PBS, incubated with 10 μg/ml of Hoechst 33342 for 45 min at 37 °C, and then examined using a flow cytometry (BD Biosciences, BD FACSDiva, USA) to assess the cell cycle distribution.

Curcumin loading efficiency (%)= Total amount of curcumin − amount of free − Cur in supernatant Total amount of curcumin

× 100

2.5. Curcumin release profile (In vitro study) The amount of curcumin released from PEG-MSNPs-Cur was determined by a dispersion technique using two different release media at pH values (pH 7.4 and 5.5). Equal amounts of PEG-MSNPs-Cur (3 ml) were poured into two separate dialysis bags (MWCO = 12 KDa), one of which was immersed in a phosphate buffer saline (pH 5.5), while the other was immersed in a phosphate buffer solution (pH 7.4). The two dialysis bags were then shaken (100 rpm at 37 °C) for 9 days under dark conditions (Sigma, Laborzentrifugen, Germany). At scheduled time intervals (0.5, 1, 2, 3, 18, 24, 48, 72, 96, and 196 h), 3 ml of the solution was withdrawn from each release media, and then the curcumin concentration was quantified spectrophotometrically at 426 nm. To maintain a constant volume, the withdrawn amount was returned, after each measurement, to the release media to keep a constant cumulative release percentage. All measurements were carried out in triplicate. The concentrations of the released curcumin were calculated from the calibration curve, after which, the cumulative release (CR %) was quantified as follows:

CR (%) =

2.7. In vivo studies 2.7.1. Animals and treatment protocols design 2.7.1.1. All animal experiments are described in (S2). The anticancer activity for both free-Cur and PEG-MSNPs-Cur, as chemopreventive and therapeutic agents, were assessed in vivo by measuring tumor growth inhibition. For this purpose, two treatment protocols were administrated: Tumor Chemoprevention Protocol (TCP) and Tumor Reduction Protocol (TRP). The design of the two treatment protocols are described in (S2). 2.7.2. Statistical analysis All data were calculated and displayed as average ± SD. The values: *P ≤ 0.05, *P ≤ 0.01, **P ≤ 0.001, and ***P ≤ 0.0001 were considered statistically significant compared to control. Experimental differences were tested for statistical significance using ANOVA one way. A P value of < 0.05 was considered significant.

Amount of curcumin released × 100 Total amount of curcumin

2.6. In vitro studies

3. Results and discussion

2.6.1. Cell culture Human cervical cancer cell line (HeLa, ATCC, USA) and human liver cancer cell line (HepG2, ATCC, USA) were kindly obtained from the stem cells unit at King Fahad for Medical Research Center (KFMRC). Both HepG2 and HeLa cells were cultured separately in DMEM supplemented with 10% fetal bovine serum (FBS) and 2% penicillin streptomycin for 24 h (at 37 °Cand humidity 5% CO2) [NuAire, CO2 incubator, USA]. After 24 h, the cells had become attached to the bottom of the flask and the media was discarded. The cells were then washed with PBS to remove the excess media. After that, trypsin was added and the trypsinized cells was examined under an inverted light microscope (OLYMPUS, inverted system microscope, Japan) to confirm that the cells were floating and alive. For all in vitro experiments, the harvested cells were resuspended in fresh media prior to plating.

3.1. Characterization of the prepared nanoparticles TEM analysis showed the monodispersity of MSNPs as shown in Fig. 1A. MSNPs were found to have a fine, smooth, and spherical shape as shown in Fig. 1B. Moreover, TEM images revealed well-arranged pore like channels on the surface of MSNPs. As depicted in our previous study, XRD pattern of MSNPs demonstrated the well-arranged pores of MSNPs [33]. DLS measurements showed the mean size distribution of PEGMSNPs-Cur which was 184.6 ± 13.69 nm, PDI value 0.516 Fig. S3. This size facilitates the passage of PEG-MANPs-Cur through tumor leaky vasculature enabling its internalization into cancerous cells [42]. The zeta Potential value of the blanked MSNPs was 3

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Fig. 1. TEM images of MSNPs and magnified MSNPs(A&B). (C) Zeta potential values of MSNPs, MSNPs-Cur, and PEG-MSNPs-Cur.(D) Cur release profile from PEGMSNPs-Cur at different pH values (7.4 and 5.5).

biocompatibility, prolongs the circulation time of the nanoformulation in blood, and prevents their phagocytosis by the reticuloendothelial system (RES) [48,49].

38.3 ± 6.09 mV, after curcumin loading MSNPs-Cur it became 36.2 ± 3 mV, and for PEG-MSNPs-Cur it was 20.8 ± 4.28 mV (Fig. 1C). The decrease in positivity of MSNPs after curcumin loading is attributed to the negative nature of curcumin. The further reduction of positivity to 20.8 mV is due to the PEG coating on the surface of MSNPsCur. These results demonstrate the high stability of PEG-MSNPs-Cur. The high positivity or negativity of the zeta potential value increases the repulsion force between the particles thus providing high stability [43]. FTIR analysis confirmed the successful preparation steps. Fig. S4 shows the FTIR spectra (4000 cm−1 to 400 cm−1) for Cur, MSNPs, MSNPs-Cur, PEG, and PEG-MSNPs-Cur. In Fig. S4(a), the characteristic bands of curcumin appeared at 1626.75, 1598.84, and 1502.54 cm−1 assigning C‒O stretching, C=O vibration of conjugated ketone, and the phenol group, respectively. The peak at 3503.45 cm−1 represents curcumin's –OH stretching bond (phenol hydroxyl group). In Fig. S4(b), the FTIR spectrum of MSNPs showed an asymmetric stretching band of 3D Si‒O‒Si at 1230.9 cm−1 which is assigned to the fine pore structure and high specific surface area of MSNPs, this is consistent with XRD pattern previously measured in our study [33]. While, the asymmetric and symmetric bands of Si‒O were observed at 1057.32 cm−1 and 796.23 cm−1, respectively. The FTIR spectrum of MSNPs-Cur revealed a decrease in the intensity for all the characteristic peaks of both curcumin and mesoporous silica nanoparticles (Fig. S4(c)). Additionally, the 3503.45 cm−1 peak of curcumin became broader and shifted to a lower wave number. Moreover, the characteristic peaks of curcumin slightly shifted. This confirms the successful adsorption of curcumin molecules in the MSNPs matrix through the formation of electrostatic interactions without altering their individual intrinsic identity. The above results are in agreement with previous studies [44–47]. Fig. S4(d) showed the characteristic peaks of PEG, which appear at 2882.16 and 1094.97 cm−1 indicating C‒H symmetric stretching and C‒O stretching. These peaks and all PEG's peaks are still present with a slight shift after MSNPs-Cur PEGylation (Fig. S4(e)). This enhances curcumin

3.2. Curcumin loading efficiency The total amount of curcumin-loaded in the pores of mesoporous silica nanoparticles MSNPs was about 92.4%.

3.3. Curcumin release profile (In vitro study) The behavior and amount of cumulative Cur released from PEGMSNPs-Cur were assessed by measuring the concentration of Cur released at different pH values 7.4 and 5.5, which simulate blood physiological environment and acidic tumor microenvironment, respectively. As illustrated in Fig. 1D, at pH 7.4, the amount of Cur released from PEG-MSNPs-Cur throughout 192 h was about 1% indicating the very slow rate of Cur release under physiological conditions. This might be attributed to the maintenance of the electrostatic interaction between MSNPs and loaded Cur at pH 7.4. This in turn protects curcumin from degradation and prolongs circulation time. Meanwhile, in the acidic medium (pH 5.5), Cur release was dramatically accelerated, the amount released was about 52% throughout the study period. This was attributed to the breakdown of the bond between Cur and MSNPs, which maximizes Cur release and concentration in tumor microenvironment. Furthermore, the high release rate of Cur might be due to the collapse of PEG in the acidic pH [50–52]. Accordingly, the prepared PEG-MSNPs-Cur is pH-responsive providing the controlled release of the drug in tumor microenvironment. It is worth noting that Cur release profiles are both pH and time dependent.

4

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Fig. 2. Fluorescence microscopic images: HepG2 cells treated with free Cur (a-d) and PEG-MSNPs-Cur (f-i) respectively at different incubation times 0.5, 6, 24, and 48 h. Hela cells treated with free Cur(k-n) and PEG-MSNPs-Cur(p-s) respectively, at different incubation times 0.5, 6, 24, and 48 h. Column 5 represents the untreated cells(control) for HepG2 (e&j) and HeLa (o&t).

retention of the Cur-nanocarrier inside cancerous cells as well as the sustained release of curcumin from PEG-MSNPs-Cur (Fig. 2c and d). During the same time intervals (24 and 48 h) cells incubated with freeCur showed negligible fluorescence (Fig. 2h and i) indicating rapid clearance of free-Cur from cancerous cells. Similarly, for HeLa cells, fluorescence images reveled nearly the same behavior as that of HepG2 cells (Fig. 2g and q). In the uptake study, Fig. 2, the HepG2 cells treated with the PEGMSNPs-Cur showed an increase in the fluorescence from 0.5 h to 6 h, which then decreased in the 6 h–24 h period, and then raised again from the 24 h–48 h. This phenomenon may be attributed to the quenching effect of high curcumin accumulation in the first 6 h [53]. It is worth mentioning that the cellular uptake and accumulation of PEG-MSNPs-Cur is cell type dependent, as the HeLa cells showed rapid uptake in the first half an hour post incubation (Fig. 2p), whereas, the HepG2 cells showed mild uptake in the same period (Fig. 2f). The intracellular accumulation for HepG2 at time points 24 h and 48 h post incubation was mild (Fig. 2r and s), whereas, the intracellular accumulation for the HeLa cells during the same period was somewhat less. The above results are in agreement with previous studies [15,47,52,54,55]. Both untreated HepG2 and HeLa cells (control) are

3.4. In vitro studies 3.4.1. Cellular uptake and intracellular accumulation In vitro cellular uptake and intracellular accumulation for both Cur and PEG-MSNPs-Cur by HepG2 and HeLa cells were examined at the specified time intervals of (0.5, 6, 24, 48 h) using fluorescence microscopy. HepG2 cells were incubated separately with PEG-MSNPs-Cur as well as free-Cur for 0.5 h, a weak fluorescence was observed indicating low cellular uptake (Fig. 2a and f). After extending the incubation time to 6 h, the intracellular accumulation decreased for the cells treated with free-Cur while those treated with PEG-MSNPs-Cur showed strong fluorescence, reflecting the high accumulation of nanocarriers inside cancerous cells (Fig. 2 b and g). The rapid clearance of Cur out of HepG2 cells after 6 h incubation is attributed to the hypophobic nature of Cur (Fig. 2b). In contrary, the retention of Cur loaded nanoparticles (PEG-MSNPs-Cur) in HepG2 cells reflects the enhancement of Cur bioavailability (Fig. 2g). Interestingly, during the first two days post incubation (24 h and 48 h), cells treated with PEG-MSNPs-Cur remained showing high fluorescence intensity. This demonstrates the accumulation and 5

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Fig. 3. Cytotoxicity of free-Cur and PEG-MSNPs-Cur (36, 45, 54, 72, 90, 108 μg/ml) against HepG2 and HeLa cells for: (A) 24 h incubation time and (B) 48 h incubation time.

HepG2 and HeLa cells treated with PEG-MSNPs-Cur (at the two incubation times 24 and 48 h) showed similar morphological changes that characterize cell apoptosis, such as rounding, floating, cells shrinkage, contact loss, as well as spindle shape distortion. Areas of dark aggregates were also observed indicating cellular internalization of PEG-MSNPs-Cur (Fig. 4C–F). These results confirmed that the curcumin nanocarrier facilitates the transfer of the hydrophobic drug (curcumin) into cancer cells, which in turn, induces apoptosis. Additionally, the surface coating of MSNPs with the PEG shell increases the biocompatibility of the nanocarrier thus providing a higher therapeutic efficacy [31]. In contrary, for HepG2 and HeLa cells treated with free-Cur (at the two incubation times 24 and 48 h), the microscopic images revealed that viable cancer cells were predominate (Fig. 4G–J). This is attributed to the low bioavailability and poor solubility of curcumin that hinders its cellular uptake and minimizes its therapeutic index [57–59].

shown in Fig. 2 (Column 5). 3.4.2. Cytotoxicity of free-Cur and PEG-MSNPs-Cur Using the WST-1 assay, the antitumor activity of PEG-MSNPs-Cur against liver cancer cells (HepG2) and cervical cancer cells (HeLa) was assessed and compared with that of free-Cur. Both HepG2 and HeLa cells were incubated separately for 24 and 48 h with different concentrations of PEG-MSNPs-Cur and their equivalent doses of free-Cur ranging from 36 to 108 μg/ml. It was obvious from the cytotoxicity results that all the selected PEG-MSNPs-Cur concentrations exerted high cytotoxic effects on both HepG2 and HeLa cells. At 36 μg/ml Cur concentration, PEG-MSNPs-Cur showed cell viability less than 10% for HepG2 cell and less than 30% for HeLa cell after 24 h which then considerably decreased to about 7% for both cancer cell types after 48 h. While for the cells treated with freeCur with the same concentration (36 μg/ml), the minimum cell viability did not exceed 76% after 48 h. At the highest Cur concentration 108 μg/ ml, PEG-MSNPs-Cur caused profound cytotoxicity as the cell viability achieved about 3% (Fig. 3A and B). The in vitro cytotoxicity assay plays an important role in evaluating the effective therapeutic dose that would prompt a significant cancer cell death. The results demonstrated that, 24 h post incubation, the half maximal inhibitory concentration IC50 for both HepG2 and HeLa cells treated with PEG-MSNPs-Cur were 20 and 28 μg/ml respectively. As depicted in Fig. 3A and B, PEG-MSNPs-Cur suppressed the proliferation of HepG2 and HeLa cells significantly in a dose and time dependent manner. In contrast, free-Cur did not induce such a high level of cytotoxicity as the maximum induced cytotoxicity was 35%. The marked cytotoxicity of PEG-MSNPs-Cur against HepG2 and HeLa cells was attributed to the polymeric coat of MSNPs which greatly facilitates the cellular uptake and intracellular accumulation of curcumin loaded nanoparticles within tumor cells [32]. Also, the acidic pH responsive release offered by the nanoformulation induced curcumin sustain release, in tumor microenvironment, over 48 h incubation time thus achieving such high therapeutic index. Our results are in agreement with previous studies [29,30,32,56]. Accordingly, the developed PEG-MSNPs-Cur enhances curcumin biocompatibility and bioavailability.

3.4.4. Cell cycle arresting assay For analyzing the distribution of the cell cycle, a flow cytometric experiment was conducted to assess the anticancer effect of PEGMSNPs-Cur compared with free-Cur for the progression of HepG2 cell cycle. In Fig. 5A, the untreated HepG2 cells (control) showed a normal proliferation rate at all cell cycle phases. Upon treatment with free-Cur, most of HepG2 cells were arrested early at S-phase and successively at G2/M-phase (Fig. 5B). As illustrated in Fig. 5C, for HepG2 cells treated with PEG-MSNPs-Cur, the cell cycle was nearly blocked at all phases inducing a high percentage of apoptosis. The higher therapeutic efficacy of PEG-MSNPs-Cur over that of free-Cur is due to the rapid internalization and accumulation of the nanocarrier inside tumor cells. Furthermore, the sustained release of curcumin over a 24 h incubation period is considered as the main factor for cell cycle arrest (Fig. 5D) [60,61]. 3.5. In vivo studies 3.5.1. Tumor growth inhibition A schematic diagram of the design of the two treatment protocols are presented in Fig. 6A.

3.4.3. Morphological examination of cancer cells using inverted microscopy The above cytotoxicity results were confirmed by examining the cells morphology of untreated and treated cancerous cells using an inverted light microscope. Fig. 4A and B show the regular spindle shape of untreated HepG2 and HeLa cells (control), respectively.

3.5.1.1. Therapeutic efficacy of the Tumor Chemoprevention Protocol. At the end of TCP, the amount of collected EAT for the untreated control group TCPcon (received PBS) was 12.5 ml (Fig. S5). Whereas in the two treated groups TCPCur (treated with free-Cur) and TCPPEG-MSNPs-Cur (treated with PEG-MSNPs-Cur), the collected EAT volumes were 6

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Fig. 4. Inverted Microscopic images for: HepG2 (left column) and HeLa cancer cells (right column); (A & B) untreated HepG2 and HeLa cells respectively (control), (C&D) HPG2 and HeLa cells treated with PEG-MSNPs-Cur for 24 hrespectively, and(E & F) HPG2 and HeLa cells treated with PEG-MSNPs-Cur for 48 hrespectively. (G & H)HepG2 and HeLa cells treated with free-Cur for 24 h and (I & J) 48 h, respectively. 20 x

about 6.5 and 2.5 ml, respectively (Fig. S5). Relative to the TCPcon group, the TCPPEG-MSNPs-Cur group showed significant tumor growth inhibition ~80% and for the TCPCur group, tumor growth inhibition was ~48% (P < 0.0001) (Fig. 6B). The results demonstrated that, PEGMSNPs-Cur has higher chemoprevention and therapeutic efficacy over free-Cur. This is because of the biocompatible smart nanohybrid silica materials that potentiate sustain curcumin release out of MSNPs pores at tumor site. PEG-MSNPs-Cur enhances Cur bioavailability and therapeutic efficacy as well as preventing the proliferation of EAT considerably by arresting the cancer cell cycle [31].

Fig. 5. Flow cytometric analysis for: (A) untreated HepG2 cells (control); (B) HepG2 cells treated with free-Cur; and (C) HepG2 cells treated with PEGMSNPs-Cur; at a concentration of 72 μg/ml for 24 h incubation. (D) Representative histogram of cell cycle distribution for: (a) untreated HepG2 cells (control), (b) HepG2 cells treated with free-Cur, and (c) HepG2 cells treated with PEG-MSNPs-Cur; 24 h post incubation.

3.5.1.2. Therapeutic efficacy of the Tumor Reduction Protocol. At the end of TRP, the collected volume of EAT was 20 ml for the untreated control group TRPcon (received PBS), while in the two treated groups TRPCur (treated with free-Cur) and TRPPEG-MSNPs-Cur (treated with PEG-MSNPsCur) the volume was about 14 and 10 ml, respectively, (Fig. S6). Thus, the percentage of tumor growth inhibition for the TRPPEG-MSNPs-Cur and TRPCur groups was about 50 % and 30 %, respectively, compared with the untreated group TRPcon (P < 0.0001) (Fig. 6C). Based on the above results, the TCP showed a higher therapeutic index compared with the conventional TRP. The administration of freeCur or PEG-MSNPs-Cur before tumor induction significantly reduced tumor growth, thus indicating the successful chemoprevention mode in

comparison with the conventional treatment protocol (TRP). Images of mice at the end of the two treatment protocols are shown in Fig. 7. 3.5.2. Angiogenesis evaluation Proliferation of cancer cells is accompanied by the formation of new blood vessels in a process known as angiogenesis. Angiogenesis ensures enough nutrition and oxygen delivery that is necessary for cancer growth [62,63]. The antiangiogenic effect of PEG-MSNPs-Cur compared to free-Cur was evaluated for the two cancer treatment protocols TCP and TRP. Fig. 8 A and D revealed a large number of blood vessels (angiogenesis) in the inner peritoneal lining of the untreated groups 7

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Fig. 6. (A) schematic diagram representing the time table for TCP&TRP. (B) Images of the collected amounts of EAT at the end of the TCP:TCPcon (untreated group), TCPCur(free-Cur treated group), and TCPPEG-MSNPs-Cur(PEG-MSNPs-Cur treated group). (C)Images of the collected amounts of EAT at the end of the TRP: TRPcon(untreated group),TRPCur (free-Cur treated group), and TRPPEGMSNPs-CurPEG-MSNPs-Cur treated group. Data is expressed as average ± SE (n = 6).

Fig. 8. Antiangiogenesis effect for: (B) free-Cur; (C) PEG-MSNPs-Cur compared with (A) the control at the end of TCP. Antiangiogenesis effect for: (E)free-Cur; (F) PEG-MSNPs-Cur compared with (D)control at the end of TRP.

(TCPcon and TRPcon) indicating the large demand of blood supply needed for the high growth rate of cancer cells. For the groups treated with free-Cur, the angiogenesis amount partially decreased reflecting the limited anticancer activity for Cur due to its low bioavailability (Fig. 8B and E.) On the contrary, the antiangiogenic effect of the PEGMSNPs-Cur markedly minimized the formation of new blood vessels contributing to the significant inhibition of tumor growth Fig. 8C and F compared to those treated with free-Cur (Fig. 8B and E). In conclusion, for the two suggested protocols, treatment with free-

Fig. 7. Images for untreated and treated tumor-bearing mice at the end of the two administrated treatment protocols TCP and TRP. Top row: mice of TCP: (A) untreated mice (control) [TCPcon]; (B) free-Cur-treated mice [TCPCur]; and (C) PEG-MSNPs-Cur-treated mice [TCPPEG-MSNPs-Cur]. Bottom row: mice of TRP: (D) untreated mice (control) [TRPcon]; (E) free-Cur-treated mice [TRPCur]; and (F) PEG-MSNPs-Cur-treated mice [TRPPEG-MSNPs-Cur].

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Table 1 Biochemical parameters for the untreated mice (TCPcon and TRPcon), mice treated with free-Cur (TCPCur and TRPCur), and mice treated with PEG-MSNPs-Cur (TCPPEGMSNPs-Cur and TRPPEG-MSNPs-Cur) at the end of the two administrated treatment protocols TCP and TRP, respectively.

All parameters are expressed as average ± SE (n = 3). For TCP, (**P < 0.01; *P < 0.05; ***P < 0.0001 vs. control). For TRP, (*P < 0.05 vs. control). The statistical significant differences were determined using one-way ANOVA.

Fig. 9. Histopathological examination of H&E stained sections for: Column 1: heart; Column 2: liver; Column 3: kidney; Column 4: spleen. Row 1: control (from untreated group); rows 2& 3: for TCP, stained sections from mice groups treated with Cur and PEG-MSNPs-Cur respectively; rows 4 &5: for TRP, stained sections from mice groups treated with Cur and PEG-MSNPs-Cur respectively.

angiogenesis. Owing to the following: (i) using nanohybrid mesoporous silica as drug delivery system protected the hydrophobic curcumin and enhanced its bioavailability in agreement with the study of C. Chen 2018, thus potentiating a chemoprevention effect against cancer growth [64], (ii) the repeated dose administration on alternative days exploited the advantageous sustained release of Cur from the MSNPs in

Cur showed mild therapeutic efficacy represented in the reduction of EAT proliferation and angiogenesis compared with PEG-MSNPs-Cur. This mild reduction was attributed to the repeated administration of doses of Cur, which overcame Cur: low bioavailability, fast degradation, and rapid systemic elimination. Meanwhile, treatment with PEGMSNPs-Cur showed significant inhibition of EAT proliferation and 9

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Appendix A. Supplementary data

an acidic tumor microenvironment achieving high Cur concentration in blood and thus offering marked tumor growth suppression [56]. The suggested TCP is promising, according to the distinct in vivo results which may open new avenues for future cancer therapeutics.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.micromeso.2019.06.002. References

3.5.3. Toxicity assessment At the end of the two treatment protocols, blood samples were withdrawn from the mice of all the experimental groups. Serum biochemical analysis was then conducted to assess the toxicity of the administrated doses of free-Cur and PEG-MSNPs-Cur on the vital organs. For TCP, the two treated groups TCPCur and TCPPEG-MSNPs-Cur showed a significant increase in their serum alanine transaminase (ALT) levels 34.5 ± 2.30 U/L and 29.63 ± 2.30 U/L, respectively, compared with the untreated group TCPcon 27.67 ± 2.30 U/L (Table 1). Also, there were marked increases in uric acid (UC) levels for the TCCur group compared with the untreated group (Table 1). Meanwhile, the other biochemical parameters aspartate transaminase (AST) and creatinine (CRE) remained unchanged indicating no toxic effects on kidney and liver. As indicated in in vivo results, the surface modified nanoformulation enhanced curcumin therapeutic efficacy against cancer with minimal collateral damage to healthy tissues [65]. For the TRP, the AST level significantly increased in the TCPEG-MSNPsCur group compared with the untreated group TRPcon. For the two treatment protocols (TCP and TRP), the alterations of some biochemical indices might be attributed to the repeated doses which induce temporal disturbance in the performance of certain body organs. Such disturbances will be recovered after the termination of the treatment protocol. Our interpretation is based on the following histopathological examination, which showed no histological alternation in kidney and liver at the end of the treatment protocols.

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3.5.4. Histopathological examination The histopathological examination was performed to assess the effect of the two administrated drugs (free-Cur and PEG-MSNPs-Cur) on the vital organs of mice at the end of the two treatment protocols. For the six experimental groups (treated and untreated) the heart muscle, liver, kidney, and spleen were collected, fixed, H & E stained, and then examined under a light microscope. Among all the experimental groups, the macroscopic examination of the four organs showed normal morphological shape with no defects or abnormalities indicating intact cells appearance (Fig. 9). These results indicated that the application of free-Cur and PEGMSNPs-Cur as anticancer drugs are nontoxic and safe on normal healthy tissue. Moreover, the repeated doses and accumulative concentration of the two drugs were non-lethal (no mice died during the treatment protocols) and did not exhibit any toxic effect on mice generally.

4. Conclusion In conclusion, our findings demonstrated that mesoporous silica nanoparticles are a promising nanocarrier for the enhancement of curcumin bioavailability and its protection from premature degradation. The results revealed that the curcumin loaded nanoparticle potentiates tumor growth inhibition significantly, thus it is considered as an efficient chemopreventive and therapeutic agent. Accordingly, the promising curcumin loaded mesoporous silica nanoparticle provides a safe and alternative approach for cancer treatment allowing its entrance into the clinical phase.

Funding This work was supported by King Abdulaziz City for Science and Technology fund, Saudi Arabia [Grant 1413-37] 10

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