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Tailoring Particle Size of Mesoporous Silica Nanosystem to Antagonize Glioblastoma and Overcome Blood-brain Barrier Jianbin Mo, Lizhen He, Bin Ma, and Tianfeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11730 • Publication Date (Web): 25 Feb 2016 Downloaded from http://pubs.acs.org on February 29, 2016

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ACS Applied Materials & Interfaces

Tailoring Particle Size of Mesoporous Silica Nanosystem to Antagonize Glioblastoma and Overcome Blood-brain Barrier Jianbin Mo#, Lizhen He#, Bin Ma, Tianfeng Chen*

Department of Chemistry, Jinan University, Guangzhou 510632, China * E-mail: [email protected].

# These authors contributed equally to the work.

ACS Paragon Plus Environment


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ABSTRACT Blood-brain barrier (BBB) is the main bottleneck to prevent some macromolecular substance entering the cerebral circulation, resulting the failure of chemotherapy in the treatment of glioma. Cancer nanotechnology displays potent applications in glioma therapy owing to their penetration across BBB and accumulation into the tumor core. In this study, we have tailored the particle size of mesoporous silica nanoparticles (MSNs) through controlling the hydrolysis rate and polycondensation degree of reactants, and optimized the nanosystem that could effectively penetrate BBB and targeted the tumor tissue to achieve enhanced anti-glioma efficacy. The nanoparticle was conjugated with cRGD peptide to enhance its cancer targeting effect, and then used to load antineoplastic doxorubicin. Therefore, the functionalized nanosystem (DOX@MSNs) selectively recognize and bind to the U87 cells with higher expression level of ανβ3 integrin, sequentially enhance the cellular uptake and inhibition to giloma cells, especially the particle size at 40 nm. This particle could rapidly enter cancer cells and was difficult to be excreted outside the cells, thus leading to high drug accumulation. Furthermore, DOX@MSNs exhibited much higher selectivity and anticancer activity than free DOX and induced the glioma cells apoptosis through triggering ROS overproduction. Interestingly, DOX@MSNs at about 40 nm exhibited stronger permeability across the BBB, and could disrupt the VM-capability of glioma cells by regulating the expression of E-cadherin, FAK and MMP-2, thus achieving satisfactory anti-glioblastoma efficacy and avoiding the unwanted toxic side effects to normal brain tissue. Taken together, these results suggest that, tailoring the particle size of MSNs nanosystem could be an effective strategy to antagonize glioblastoma and overcome BBB.

KEYWORDS: particle size, glioblastoma, BBB, apoptosis, mesoporous silica nanosystem


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INTRODUCTION The incidence and mortality of cancer patients are increasing gradually, and thus cancer is becoming one of the most devastating diseases in all over the world.1-2 Specially, glioma is the most lethal type of cancer with extremely poor prognosis and high relapse because of its highly infiltrating property.3-4 The invasive glioma cells rapidly infiltrate and hide in the areas of the brain, which make it completely impossible to be completely eliminated by traditional surgical methods of surgery and radiotherapy.5-6 At the same time, the failure of chemotherapy in the treatment of glioma mainly owing to the inability of chemotherapeutic drugs to penetrate across the blood-brain barrier (BBB) into the brain tissue.7-8 BBB plays an important role to separate the blood and brain tissue and selectively stop some toxic macromolecular substances entering the cerebral circulation.9-10 The other obstacle to cure the glioma is the vasculogenic mimicry (VM) capacity of glioma cells.11 VM was described as the functional vascular channels only formed by tumor cells, which was used to provide nutrients for the glioma cells during the early growth of cancer or the regeneration of residual cells after cancer therapy.12-14 Therefore, VM-capability of glioma cells is responsible for the invasion and high drug resistance of brain cancer.15-16 Till now, the studies of novel drugs to overcome the obstacles of BBB and VM have aroused widespread attention. Recently, Elisa Salvati et al. reported the amyloid-β-targeting liposomes decorated with anti-transferrin receptor antibody as a suitable nanosystems to overcome blood-brain barrier and target the Alzheimer’s disease.17 Thomas et al. also investigated a new peptide-paclitaxel conjugate to be easily taken into brain.18 Therefore development of an efficient anticancer nanodrugs to enhance the permeability across the BBB, simultaneously destroy their VM-capability becomes an impending mission. As an interdisciplinary technology, cancer nanotechnology shows enormously potential applications in cancer prevention, targeted therapy and molecular imaging.19-22 Through carrying small molecular drugs, cancer targeted nanodrug delivery systems are expected to lessen the toxic side effect and enhance their anticancer activity, thus have shown excellent potential application in the cancer



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therapy.23-25 There are a variety of biocompatible nanodrug carriers have aroused a great interest in cancer therapy, such as silica nanoparticles,26-27 nanoliposome28 and magnetic nanoparticles.29 In recent years, various nanodrugs have been reported to solve the problems confronted by the conventional drugs in clinic, such as the high side effects, low selectivity and the inability of transport across the BBB. Among the fast development of nanodrugs, mesoporous silica nanoparticles (MSNs) reveal obvious superiorities over other nanomaterials due to low toxicity, the controllable size of particle and pore morphologies, and facile surface decoration, which are favorable for the rational design to solve the problems faced in the cancer therapy.30-34 For example, Neetu Singh et al. have reported a bioresponsive MSNs nanosystem with temperature sensitivity to control the release of drugs, prolong the blood circulation and depress its toxic side effects.35 Furthermore, Jie Lu et al. also designed and synthesized mesoporous silica nanoparticles-based drug delivery system with folate modification, which exhibited the significant tumor-suppression effects to two different human pancreatic cancer xenografts on different mouse species.36 At the same time, they also found the highlight attractive features of MSNs nanodrugs with biocompatibility, renal clearance and high efficacy for delivering anticancer drugs. As the failure of chemotherapy to glioma is because of the inability of drugs to transport across the BBB and arrive at the brain tumor tissue.37 Accordingly, development of a novel nanodrug simultaneously to realize the targeted recognition to glioma and enhance the permeability across the BBB is practical and urgently needed. Recently, studies have focused on the cationic homopolymers and peptide-decoration to enhance the permeability and internalization of nanosystems in overcoming of glioblastoma.38-39 For instance, Li et al. have synthesized two multifunctional liposomes with MAN-TPGS1000 and DQA-PEG2000-DSPE which found that their transport mechanism across the BBB was associated with glucose transporters and electric charge-based interactions-mediated endocytosis, respectively.9 Park et al. also found that poly(mannitol-co-PEI) modified transporter could significantly enhance the BBB permeability during the therapeutics in Alzheimer's disease.40 As we all know, the size of nanoparticles also affect the endocytosis by cells. The relationship between


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the size of the nanoparticles and their permeability across the BBB and the inhibition efficacy to glioblastoma remains elusive. Thereupon, an appropriate size of MSNs drug delivery system for anti-glioblastoma should be groped urgently. In this study, we have tailored the size of MSNs nanoparticles, then loaded with DOX and modified by cancer-targeted polymer of PEI-cRGD to enhance the anti-glioblastoma effects. Among the different-sized DOX@MSNs, the particle size at about 40 nm exhibited the highest anticancer activity to glioma, witch because of the rapid internalization in cancer cells and difficult discharge outside the cells, finally leading to the highest cellular uptake. These nanosystems could permeate across the BBB, disrupt the VM-capability of glioma cells, cut off the supply of nutrition necessary for the glioma cells growth and regeneration, and kill glioma cells selectivity, thus achieving satisfactory anti-glioblastoma efficacy and avoiding the unwanted toxic side effects to normal brain tissue. Taken together, these results suggest that, tailoring the particle size of MSNs nanosystem could be an effective strategy to antagonize glioblastoma and overcome BBB.

EXPERIMENTAL SECTIONS Materials and cell lines Tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), triethanolamine (TEA), diethanolamine (DEA), polyetherimide (PEI, Mw=10,000), and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Chemistry Co., Ltd. The cRGD (cricoids Arg-Gly-Asp-Phe-Lys) peptide was purchased from Gier Biochemistry CO, LTD. (shanghai, china). Human glioma cells lines, including U87 and U251 cells, mouse glioma cells C6 cells and human normal brain colloid cells (CHEM-5) and human brain microvascular endothelial cells (HBMEC) were purchased from American Type Culture Collection (Manassas, VA). All the cells were cultured in DMEM media with 10% fetal bovine serum, 100 units/ml of penicillin and 50 units/ml of streptomycin at



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37 ℃ in CO2 incubator (95% relative humidity, 5% CO2).

Synthesis of different-sized DOX@MSNs nanosystems According to the previously reported, the MSNs nanoparticles were synthesized with some changes.32 Curtly, 1.0 g of the CTAB were dissolved in 20 ml water and stirred for 30 min. Subsequently, the different addition of DEA were appended to the CTAB solution and churned at 95℃ for 1 h. And then 1.5 ml (about 1.4 g) of the TEOS was added into the above solution dropwise and then stirred for another 1h. After that, the products could be collected by centrifugation, and then refluxed in methanol/HCL for 12 h to get rid of the template of CTAB to obtain the different-sized MSNs nanoparticles. Then we changed the template to CTAC and base catalyst of TEA to control the sizes of MSNs particles. To obtain the different-sized DOX@MSNs nanosystems, we chose three different particle sizes of MSNs (20, 40 and 80 nm) as the carriers for DOX, and modified with PEI-cRGD. Briefly, 100 mg of different-sized of MSNs were suspended in 10 ml of DOX (1 mg/ml) and stirred for 24 h at room temperature, respectively. Then we collected these nanoparticles by centrifugation and continue stirred with the before prepared PEI-cRGD solution for another 24 h. Finally, the different-sized DOX@MSNs nanosystems were collected by centrifugation and freeze drying. For preparation of the PEI-cRGD, 25 mg of PEI was dissolved into 5 ml of Milli-Q water and added 10 mg of EDC into the solution, stirred for 2 h. The 5 mg/ml of cRGD peptide (2 ml) solution was then added into the mixed solution and stirred for another 24 h. After that the PEI-cRGD solution was dialyzed against Milli-Q water for 6 h to remove the excess cRGD and EDC.

Characterization of DOX@MSNs nanosystems MSNs nanoparticles solution was dispersed onto holey carbon film on copper grids, and then conducted by transmission electron microscopy (TEM) on Hitachi (H-7650) operated at an acceleration voltage of 80 kV. The size distribution and Zeta potential of MSNs and DOX@MSNs were measured on Nano-ZS instrument (Malvern Instruments Limited). Through a NOVA 4200e surface area analyzer (Quan-tachrome), nitrogen adsorption-desorption isotherms were acquired at -196 °C under continuous adsorption conditions. FT-IR spectrometer (Equinox 55, Bruker)


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was used to analyze the difference of FT-IR spectra of the samples in the range 4000-400 cm-1.

MTT assay Cells (2×104 cells/ml) were seeded in 96-well tissue culture plates for 24 h, and then incubated with the different-sized DOX@MSNs nanoparticles at different concentrations for 72 h. After incubation, 30 µl/well of MTT solution (5 mg/ml PBS) was added and incubated for 4 h. And then, the culture medium was removed and replaced with 150 µl/well of DMSO. The color intensity of the DMSO solution was measured at 570 nm using microplate spectrophotometer (SpectroAmaxTM 250) to calculate the cell viability.

Cellular uptake of DOX@MSNs For quantifying cellular uptake of DOX@MSNs and free DOX in glioma and normal cells, cells were cultivated into 96-well plates at 10,000 cells/well (0.1 ml) for 24 h. Free DOX and different-sized DOX@MSNs were added into each well, and the final drug concentration was 2 µM. Then, the cells were incubated at 37 ℃ for 4 h or 8 h. Finally, the medium was removed from the wells and the cells were rinsed three times with cold PBS to remove the nanoparticles outside the cells. After that, 100 µl of 0.04 M HCl/DMSO solution was appended to lyse the cells. The fluorescence intensity of drug in above lysate was measured by fluorescence microplate reader (Spectra Max M5, MD, USA). For the RGD blocking cellular uptake of different-sized DOX@MSNs, the glioma cells were pretreated with different concentration of RGD for 2 h to block the target spots on the surface of glioma cells, and then exposed to 1µM of different-sized DOX@MSNs for 8 h. Then the cellular uptake of the nanodrugs was measured as the front methods.

Drug retention of DOX@MSNs To analyze the drug retention of DOX@MSNs nanoparticles and free DOX in glioma and normal cells, the cells were seeded into 6-well plates at 400, 000 cells/well



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(2 ml) for 24 h, and added 1 µM of different-sized DOX@MSNs and DOX in DMEM solution to incubate for 6 h. Then, the medium with drug was removed from the well and the cells were rinsed with PBS three times to remove the drugs outside cells. And then, 2 ml of fresh culture medium were appended, and at specific time during incubation, 100 µl of supernatant were taken out from the well and kept in 96-well plates. Then the fluorescence intensity of all samples were determined by fluorescence microplate reader (Spectra Max M5, MD, USA) with the excitation and emission wavelengths set at 485 and 590 nm, respectively.

In vitro drug release of DOX@MSNs 10 mg of different-sized DOX@MSNs were added in 10 ml of PBS (pH 7.4) and glioma cell lysate with immobile shaking at 37℃ in a glass beaker, respectively. At specific time during incubation, 100 µl of supernatant was taken out from the beaker and the same volume of fresh PBS and cell lysate was replaced. All samples were determined by fluorescence intensity with the excitation and emission wavelengths set at 485 and 590 nm, respectively.41

Real-time living cell mornitoring Cells were seeded into 2 cm-culture dishes at the density of 80,000 cells/ml (2 ml) for 24 h, and incubated with 1 µl of DAPI at the concentration of 1 µg/ml for 30 min to stain the cell nucleus. Then the glioma cells were treated with 0.1µM of different-sized DOX@MSNs, the cells were examined using a fluorescence microscope (EVOS® FL) at specific time.42

Flow cytometric analysis Flow cytometric analysis was used to examine the cell cycle distribution in glioma cells after drug treatments.43 Cells were seeded into 6-cm culture dishes at 20,000 cells/ml (2 ml) for 24 h, and then incubated with different-sized DOX@MSNs at 12.5 nM for 24 h. After that the treated cells were collected and fixed with 70% ethanol overnight at -20℃. The fixed cells were collected by centrifugation and


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stained with propidium iodide (PI, 1.21mg/ml Tris) for 2 h in darkness. Besides, cell cycle distribution of glioma cells without any treatment was set as control group. The stained cells were analyzed with Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Cell cycle distribution was analyzed using Multi Cycle software (Phoenix Flow Systems, San Diego, CA). For each experiment, over 10,000 events per sample were recorded.

Measurement of intracellular reactive oxygen species (ROS) generation The effects of drug on the intracellular ROS generation in glioma cells were monitored by DHE-DA assay as previously reported.44 Briefly, glioma cells (2×105 cells/ml) were incubated in 96-well plates for 24 h, and then the culture medium were replaced with 100 µl of DHE/DMEM medium at 37 ℃ for 30 min, and the final DHE-DA concentration is 10 µM. Then 125 nM of different-sized DOX@MSNs and free DOX were added into the wells. The fluorescence intensity of DHE was measured by a fluorescence microplate reader (Spectra Max M5, MD, USA) with the excitation and emission wavelengths at 300 and 610 nm, which was used to express the ROS level activated by drugs in glioma cells.

Western blot analysis Total cellular proteins of glioma and normal CHEM-5 cells were obtained by incubating cells in lysis buffer (Beyotime) and protein concentrations were examined by BCA assay. The expression levels of integrin were determined by Western Blotting.45 The expression of β-actin was used as internal standard to analyze the amount of protein in each lane.

Transportation across the blood-brain barrier of DOX@MSNs To verify different-sized MSNs drug delivery system could transport cross BBB, a HBMEC/U87 co-culture as BBB model was established according to previous reports.46 Firstly, HBMEC cells were seeded on the upper side at 200, 000 cells per insert, and U87glioma cells were seeded on the basolateral compartment of the insert



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at 10, 000 cells per insert. After incubation for several days, until the resistance of HBMEC on the upper side was examined to over 250 Ω•cm2, 0.1µM of DOX@MSNs nanoparticles and free DOX were added into the upper side and incubated for 48 h. Then the drug transmittance ratio was measured by fluorescence microplate reader (Spectra Max M5, MD, USA). And we also examined the cellular uptake of the U87 glioma cells by fluorescence microscope (EVOS® FL). The percentage of surviving U87 cells was measured by MTT assay.

Destruction of brain cancer VM channels by DOX@MSNs A matrigel-based tube formation assay was used to examine the destruction efficacy of different-sized DOX@MSNs against the formation of VM channels of U87 glioma cells. Firstly, 100 µl of growth factor-reduced matrigel was added into 48-well plates, and incubated for 30 min at 37 ℃. Then, 300 µl of U87 glioma cells were mixed with different-sized DOX@MSNs (0.03 µM), and seeded in the wells at 5, 0000 cells/ml. After incubation at 37 ℃ for 48 h, the tubules in each well were imaged under a microscope.

Penetrating ability and inhibitory effects of DOX@MSNs to brain tumor spheroids The U87 tumor spheroids were formed and grown in 6-well plates plated with agarose according to previous procedures.9 Briefly, 2% (W/V) agarose solution were heated to 80 ℃ for 30 min, and then autoclaved for 30 min. Then the agarose solution was added into 6-well plates at 1 ml/well. After cooling to ambient temperature, 1 ml of U87glioma cells was seeded into each well at a density of 400, 000 cells/well. The plates were wobbled gently every 2h and incubated at 37 ℃ for 4 days to formed as the U87 tumor spheroids with diameters up to 400 mm. Whereafter, U87 glioma spheroids were treated with 1 µM of different-sized DOX@MSNs and free DOX for 12 h. To determine the penetration ability of different drug, the tumor spheroids were rinsed with PBS and scanned at the different layers from the top of the tumor spheroids to the middle using a confocal laser scanning fluorescent microscope. And


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we also measured the minor (dmin) and major (dmax) diameters of each tumor spheroid under microscope (EVOS® FL), the volume of spheroid was computed using the following formula: V= (π×d max×d min)/6 as the previous reported methods.

Statistics analysis All the data are expressed as mean ± standard deviation. Differences between the control and the experimental groups were analyzed by two-tailed Student’s t test. One-way analysis of variance (ANOVA) was used in multiple group comparisons. Statistical analysis was performed using SPSS statistical program version 13 (SPSS Inc., Chicago, IL). Difference with P

Tailoring Particle Size of Mesoporous Silica ... - ACS Publications

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