Dual-drug delivery by porous silicon nanoparticles for improved cellular uptake, sustained release, and combination therapy.

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Chang-Fang Wang a,⇑, Ermei M. Mäkilä a,b, Martti H. Kaasalainen b, Marja V. Hagström c, Jarno J. Salonen b, Jouni T. Hirvonen a, Hélder A. Santos a,⇑ a b c

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Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014, Finland Laboratory of Industrial Physics, Department of Physics and Astronomy, University of Turku, FI-20014, Finland Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland

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Dual-drug delivery by porous silicon nanoparticles for improved cellular uptake, sustained release, and combination therapy

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Article history: Received 23 September 2014 Received in revised form 12 January 2015 Accepted 16 January 2015 Available online xxxx

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Keywords: Intracellular uptake Sustained release Combination therapy Porous silicon nanoparticle Methotrexate

a b s t r a c t Dual-drug delivery of antiangiogenic drug and chemotherapeutic drug can enhance the therapeutic effect for cancer therapy. Conjugation of methotrexate (MTX) to PSi nanoparticles (MTX–PSi) with positively charged surfaces can improve the cellular uptake of MTX and inhibit the proliferation of cancer cells. Herein, MTX–PSi conjugates sustained the release of MTX up to 96 h, and the released fragments including MTX were confirmed by mass spectrometry. The intracellular distribution of the MTX–PSi nanoparticles was confirmed by transmission electronic microscopy. Compared to pure MTX, the MTX–PSi achieved similar inhibition of cell proliferation in folate receptor (FR) over-expressing U87 MG cancer cells, and a higher effect in low FR-expressing EA.hy926 cells. Nuclear fragmentation analysis demonstrated programed cell apoptosis of the MTX–PSi in the high/low FR-expressing cancer cells, whereas PSi alone at the same dose had a minor effect on cell apoptosis. Finally, the porous structure of MTX– PSi enabled a successful concomitant loading of another anti-angiogenic hydrophobic drug, sorafenib, and considerably enhanced the dissolution rate of sorafenib. Overall, the MTX–PSi nanoparticle system can be used as a platform for combination chemotherapy by enhancing the dissolution rate of the hydrophobic drug and sustaining the release of the conjugated chemotherapeutic drug. Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

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1. Introduction

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Nanomedicines have been investigated for cancer therapy for several decades, with several products in clinical trials or in the market [1]. Safe and efficient delivery of poorly-water soluble and/or low permeable chemotherapeutic drugs is still one of the main tasks to be overcome by cancer medicines. Porous silicon (PSi) nanomaterials possess high biocompatibility and have a number of unique properties that render them as potential drug delivery carriers in biomedical applications [2–12], such as increasing the dissolution rate of poorly water-soluble drugs [13], high drug loading capacity [14], and tunable surface structure for tailoring the biological activities by different surface chemistries [8,15–17]. For example, the carboxylic acid- and amine-terminated

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⇑ Corresponding authors at: Division of Pharmaceutical Chemistry and mTechnology, Faculty of Pharmacy, P.O. Box 56 (Viikinkaari 5E), University of Helsinki, FI-00014, Finland. Tel.: +358 9 19159661; fax: +358 9 19159144. E-mail addresses: chang-fang.wang@helsinki.fi (C.-F. Wang), helder.santos@ helsinki.fi (H.A. Santos).

surfaces of PSi can be used for further chemical functionalization [15,18,19]. Chemical conjugation of anticancer drugs to nanoparticles has been one of the approaches to overcome the drug solubility/permeability obstacles for the delivery of chemotherapeutics [20,21]. Methotrexate (MTX) is a folic acid analog anticancer drug [22]. MTX is able to inhibit the activity of dihydrofolate reductase (DHFR) enzyme in the cytosol of the cells, which results in the suppression of purine and pyrimidine precursor synthesis and consequently DNA biosynthesis, leading to cell apoptosis [23]. MTX has specific affinity to the folate receptor (FR) and good cellular uptake by certain cells due to the FR-mediated cellular uptake pathway [24]. Over-expression of the FR has been found in many human malignancies, especially when associated with aggressively growing tumors [25]. The cancer cells highly expressing the FR include ovarian, endometrial, colorectal, breast, lung, renal cell carcinoma, brain metastases derived from epithelial cancer, and neuroendocrine carcinoma cells [25,26]. However, MTX has low cellular uptake in receptor deficient cells [20]. As with many other chemotherapeutics, drug resistance has been an obstacle faced by patients when treated with MTX [22].

http://dx.doi.org/10.1016/j.actbio.2015.01.021 1742-7061/Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

Please cite this article in press as: Wang C-F et al. Dual-drug delivery by porous silicon nanoparticles for improved cellular uptake, sustained release, and

Q1 combination therapy. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.01.021

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One mechanism of the MTX resistance is to decrease the receptor expression and thus reduce the receptor-mediated cell uptake [27]. Cationic nanoparticles can be non-specifically up-taken in the cells through endocytosis by interacting with the negatively charged cell membrane. By chemically conjugating MTX to the cationic carriers, the cellular uptake can be increased [28–30]. Conjugation of MTX to chitosan [28], dendrimeric polyamidoamine [29,31,32], poly-L-lysine [20,30,33], targeting peptide cyclo(1,12) PenPRGGSVLVTGC [34], gelatin [35], human serum albumin [36], magnetic nanoparticles [37], quantum dots [38], multi-walled carbon nanotubes [39], and porous silica [40] have been reported to enhance the cell uptake and/or to overcome drug resistance. The conjugation was achieved by a covalent linkage formed between the carboxylic acid group of the MTX and the amine groups of the conjugated carriers. The 3-aminopropyltriethoxysilane (APTES) surface functionalized thermally carbonized PSi particles have amine-terminated groups on the surface of the PSi [19]. The conjugation of MTX to these biodegradable PSi nanoparticles with cationic charge can aid in improving the MTX cell uptake and inhibit the proliferation of cancer cells. The porous structure of MTX-conjugated PSi nanoparticles can be further used to load another hydrophobic drug for combination therapy. For example, the anti-angiogenic sorafenib is a multi-kinase inhibitor by targeting RAf kinase, platelet-derived growth factor, vascular endothelial growth factor (VEGF) receptor 2&3 kinases and c-Kit receptor [41]. Clinical studies have reported a high interindividual variability in the pharmacokinetics, clinical efficacy and adverse effects of sorafenib treatment [42]. Side effects and variation in pharmacokinetics caused by low solubility increase the risks associated with the clinical applications of sorafenib [43,44]. By enhancing the aqueous solubility of sorafenib, controllable plasma concentrations and more precise control on the effects of sorafenib can be attained, with reduced clinical risks to patients. In this work, the chemotherapeutical anticancer drug MTX was chemically conjugated to the amine-terminated thermally carbonized PSi to form MTX–PSi nanoparticles for enhancing the cell uptake and sustaining the release of MTX. Simultaneously, the hydrophobic anti-angiogenic sorafenib was loaded to the MTX– PSi to enhance the dissolution rate of this drug for combination therapy. The propose of the dual-drug delivery system developed in this work was to: (1) enhance the dissolution rate of the poor water soluble anti-angiogenic drug, sorafenib, which can inhibit the kinase of the neovascular cells on the cell membrane to slow down the tumor growth; and (2) to enhance the cellular uptake and sustain the release of the chemotherapeutical drug MTX to kill tumor cells. The conjugation of MTX, sorafenib loading to MTX–PSi nanoparticles, the dissolution profiles of MTX and sorafenib, as well as the anti-proliferation and cell apoptosis of MTX–PSi were evaluated in this study.

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2. Materials and methods

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2.1. Materials and cell culturing

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MTX was purchased from TCI (Chuo-ku, Japan). Sorafenib was obtained from LC Laboratories (Woburn, USA). All other chemicals and solvents purchased were of analytical grade from Sigma– Aldrich (USA) and used as received. Hypoxanthine–aminopterin– thymidine (50 HAT) was purchased from GibcoÒ (Carlsbad, USA). Dulbecco’s phosphate buffered saline (10 PBS) and Hank’s balanced salt solution (10 HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin (2.5%), sodium pyruvate, nonessential amino acids (100 NEAA), L-glutamine (100), penicillin–streptomycin (100) were all purchased from

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HyClone (Waltham, USA). CellTiter-GloÒ luminescent cell viability assay kit was purchased from Promega (Madison, USA). MTX was dissolved in dimethylsulfoxide (DMSO, 2 mM) as stock solution for further tests. Endothelial EA.hy926 (ATCC, USA) cells were incubated in DMEM with high glucose and 2% HAT, while brain cancer cells U87 MG (ATCC, USA) were cultured with DMEM with low glucose. Both cell lines were cultured in 75 cm2 flasks for further experiments at 37 °C with humidified atmosphere containing 5% CO2, and the DMEM was supplemented by 10% FBS, 1% sodium pyruvate, 1% NEAA, 1% L-glutamine, and 1% penicillin–streptomycin (100 IU/mL).

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2.2. Preparation of PSi nanoparticles

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Amine-functionalized thermally carbonized PSi nanoparticles were prepared by adapting the procedure as previously reported elsewhere [19]. Briefly, multilayer PSi films were produced by electrochemically etching monocrystalline p+-type Sih1 0 0i wafers of 0.01–0.02 X cm resistivity in a 1:1 (v/v) aqueous hydrofluoric acid (HF, 40%) – ethanol electrolyte as described previously [4]. The free standing films were thermally carbonized with acetylene at 500 °C and 820 °C [13], after which the obtained films were immersed into HF to generate surface silanol termination for APTES attachment, following the previously described process using a 10 v-% APTES-toluene solution [19]. The size reduction of the PSi multilayer films to nanoparticles was performed by wet-milling using a 5 v-% APTES-toluene solution as the milling liquid. After milling, the excess silane was removed by replacing the liquid and re-dispersing the nanoparticles to fresh toluene and ethanol at least 3 times using centrifugation. The final size selection of the nanoparticles was done by centrifugation.

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2.3. Conjugation of MTX to PSi nanoparticles

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MTX was conjugated to the amine-terminated PSi nanoparticles using N-hydroxysuccinimide (NHS) and 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) coupling reaction. Briefly, 2.2 mg (5 lmol) of MTX was dissolved in 1 mL DMSO with 10 mM of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, pH 5.5), 1.7 mg (15 lmol) of NHS and 13 lL (60 lmol of EDC) were added to the solution. The mixture was stirred for 30 min at room temperature to activate the carboxylic acid group of MTX. After that, 1 mg of the amine-terminated PSi nanoparticles in ethanol was added to the reaction mixture, and the pH was adjusted to 7.5 by 1 M NaOH. After 45 min reaction, the PSi nanoparticles were collected from the reaction mixture by centrifugation (Sorvall RC 5B plus, thermo Fisher Scientific, USA) at 10,000 rcf for 3 min and washed with 1 mL of DMSO, water and ethanol 3 times to obtain the MTX conjugated PSi nanoparticles (MTX–PSi), which were then re-suspended in ethanol for further use.

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2.4. Physicochemical characterization of PSi nanoparticles

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The physical parameters of the PSi nanoparticles were determined by nitrogen sorption at 196 °C using TriStar 3000 (Micromeritics Inc., USA). The specific surface area of the PSi nanoparticles was calculated using the Brunauer–Emmett–Teller theory. The total pore volume was obtained as the total adsorbed amount at a relative pressure p/p0 = 0.97. The average pore diameter was calculated from the obtained surface area and pore volume by assuming the pores as cylindrical. The qualitative analysis of the MTX–PSi nanoparticles were performed by Fourier transform infrared spectroscopy (FTIR) with a Vertex 70 FTIR spectrometer (Bruker Optics, USA) using a

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horizontal attenuated total reflectance (ATR) accessory (MIRacle, PIKE Technologies, USA). The transmittance spectra were recorded between 4000 and 650 cm1 with a 4 cm1 resolution. The amount of MTX covalently conjugated to the PSi nanoparticles was determined by elemental analysis using a Vario Micro cube CHN analyzer (Elementar Analysensysteme, GmbH, Germany). The percentages of carbon (C), hydrogen (H) and nitrogen (N) were recorded. The conjugation efficiency was calculated based on the percentage of each element and the chemical structure of each sample. The particle hydrodynamic diameter (Z-average) and the zeta (f)-potential measurements of the nanoparticles were carried out by dynamic light scattering (DLS) using a Zetasizer Nano ZS (Malvern Instruments, UK) equipped with a He–Ne laser beam (633 nm, fixed scattering angle of 173°) at 25 °C. The reported data are presented as the average of three independent measurements, i.e., the data are an average of three Z-average size values. The standard deviations were calculated based on the three Z-average size values. The morphology of the nanoparticles was observed by transmission electron microscope (TEM) (Jeol, JEM-1400, Japan) with voltage of 80 kV and magnification between 250 and 10,000. The samples were prepared by adding one drop of nanoparticle suspension to a copper grid coated with carbon and dried overnight before imaging.

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2.5. Cell uptake of nanoparticles

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Endothelial EA.hy926 cells, which have low expression of FR, and brain tumor U87 MG cells, which have high expression of FR, were used to evaluate the cell uptake efficiency of MTX–PSi by TEM. About 2 mL/well of 5 105 cells/well of EA.hy926 cells or 6 105 cells/well of U87 MG were seeded in 6-well plates containing 18 18 mm cover slip (Menzel-Gläser, Braunschweig, Germany) in each well. After reaching 80% confluency, the medium was replaced by a new medium containing 100 lg/mL of each nanoparticle. After 2 h incubation, the nanoparticle suspensions were removed and the cells were washed 3 times with HBSS (pH 7.4). 1 mL/well of 2.5% glutaraldehyde was added to the cells and incubated at 37 °C for 20 min for cell fixation, followed by washing 3 times with HBSS. Ultrathin sections of both control and exposed cells to the nanoparticles were prepared as described elsewhere [45]. The TEM images were obtained with the same parameters as described above.

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2.6. Drug loading and release

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The poorly water-soluble sorafenib was loaded to the MTX–PSi nanoparticles for combination therapy. The PSi and MTX–PSi nanoparticles were immersed in 15 mg/mL of sorafenib dissolved in acetone solution with a ratio of 1 mg of PSi nanoparticles to 1 mL of drug in solution, and stirred for 2 h at room temperature. The drug-loaded nanoparticles were collected and washed once with Milli-Q water. The loading degrees were determined by immersing 200 lg of the dual drug-loaded nanoparticles to 1 mL of acetonitrile/water mixture (42:58%, v/v) under vigorous stirring for 1 h. The released MTX fragment from the MTX–PSi nanoparticles was confirmed by a Waters ACQUITY UPLCÒ (Waters, Massachusetts, MO, USA) coupled to a WaterÒ Xevo™ quadrupole time-of flight mass spectrometer (Q-TOF MS, Waters, Massachusetts, MO,). The release profiles of MTX from the MTX–PSi nanoparticles loaded with/without sorafenib were performed at 37 ± 1 °C in sink conditions using a shaking method with a shaking speed rate of 100 rpm. The release media used were 10 mM of 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.5), which mimics the pH of the endosome inside the cells when the PSi–MTX are uptaken by the cells, and 10 mM HEPES (pH 7.4), which is the physiological

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pH condition, with/without 10% FBS. 10% FBS was added to the media for the drug release studies in order to confirm two effects. Firstly, these studies evaluate whether the protein influences the MTX release from the PSi cargo. Secondly, based on our previous study [17], sorafenib can be dissolved very fast from the PSi nanoparticles in a buffer containing 10% FBS, but is not released from the nanoparticle in a buffer without FBS. In this study, we compared the MTX release from the PSi nanoparticles with/without loaded sorafenib to confirm whether sorafenib loading in MTX– PSi nanoparticles affected the release of MTX. The dissolution profiles of sorafenib-loaded in both PSi and MTX–PSi, or free sorafenib, were performed at pH 7.4 with 10% FBS, because the purpose of this study was to prove that the solubility of sorafenib was enhanced in physiological conditions. 200 lL of samples was withdrawn from each dissolution test at predetermined time points. The collected samples were centrifuged at 13,400 rcf for 3 min and the supernatant was analyzed by HPLC as described below. The amounts of MTX and sorafenib released from the PSi nanoparticles were determined by HPLC (Agilent 1260, Agilent Technologies, USA). The chromatographic separation was achieved using a Zorbax C18 (4.6 100 mm, 5 lm) column. For MTX, the mobile phase used was composed of 90% of aqueous phase (0.1 M citric acid and 0.2 M Na2HPO4 mixed at a ratio of 2:1, pH 6.0) and 10% of acetonitrile with flow rate of 1 mL/min, while a UV detector was set at the wavelength of 302 nm. For sorafenib, the mobile phase was composed of 0.2% trifluoroacetic acid (pH 2) and acetonitrile at a ratio of 42:58% (v/v) with 1.0 mL/min flow rate and UV detector set at the wavelength of 254 nm.

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2.7. Anti-proliferation activity

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The in vitro anti-proliferation effect of the MTX–PSi nanoparticles against the cancer cells was evaluated by cell proliferation experiments. Briefly, EA.hy926 and U87 MG cells were seeded in 96-well plates at the density of 1 104 cells/well and 1.5 104 cells/well, respectively, and allowed to attach overnight. Then, the cell culture medium was replaced by 100 lL of medium containing different concentrations of MTX and MTX–PSi nanoparticles. After 24 and 48 h incubation, the amount of living cells was determined by the CellTiter GloÒ luminescence cell viability assay kit. Each experiment was performed at least in triplicate.

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2.8. MTX–PSi induced cell apoptosis

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MTX induces cell death by inhibiting the activity of DHFR, and thus, inhibiting the DNA biosynthesis. In order to confirm the bioactivity of the released MTX fragments from the MTX–PSi, 7amino-actinomycin D (7-AAD, BioLegnadÒ) labeling DNA fragmentation programed cell death experiment was performed by flow cytometry analysis. Briefly, 2 105 cells/mL/well of each cell line was seeded in 12-well plates separately. After reaching 80% confluency, the medium was replaced by a new medium containing certain concentrations of both PSi and MTX–PSi nanoparticles for 24 h incubation. After removing the nanoparticle solutions and washing the cells 3 times with HBSS (pH 7.4) to remove non-adherent nanoparticles, the cells were harvested by trypsin-ethylenediaminetetraacetic acid and kept on ice. Cells were then re-suspended in 100 lL BioLegand’s Cell Staining Buffer and 5 lL of 7-AAD (50 lg/mL) was added to each sample and incubated for 15 min at room temperature in the dark. After that, 400 lL of HBSS buffer was added and the flow cytometry experiments were performed with a LSR II flow cytometer (BD Biosciences, USA) using a FACSDiva software and a laser excitation wavelength of 488 nm. 10,000 events were collected for each sample and the data were analyzed by FACSDiva software.

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2.9. Statistical analysis

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Results of the assays were expressed as mean ± s.d. of at least three independent experiments. Student’s t-test was used to evaluate the significant differences and set at probabilities of ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 using Origin 8.6 (OriginLab Corp., USA).

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3. Results and discussion

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3.1. Characterization of MTX–PSi nanoparticles

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Nitrogen sorption results indicated that the amine-terminated PSi nanoparticles retained a porous structure according to the shape of the isotherm. The calculated specific surface area was 301 ± 16 m2/g with a pore volume of 0.60 ± 0.01 cm3/g, and the average pore diameter of 8.0 ± 0.4 nm. The zeta (f)-potential of the PSi nanoparticles obtained from electrophoretic mobility measurements was +40.6 mV. The conjugation of MTX to PSi was based on the NHS/EDC coupling reaction between the carboxylic acid group of MTX and the free amine groups of the PSi nanoparticles to form an amide bond (Scheme 1). According to ATR–FTIR (Fig. 1a), the exhibition of two characteristic bands of MTX–PSi corresponded to m(NC@O) (amide I) and d(CNAH) (amide II) vibrations at 1650 and 1540 cm1, respectively, which indicated the formation of an amide bond between MTX and the nanoparticle. The strong peak showed at 1610 cm1 represented the typical NAH bending vibrations of primary amine in MTX. The peak 1450 cm1 was assigned to CH2 bending of MTX. All these indicated that MTX was conjugated to PSi nanoparticles. From the MS measurements of the MTX release solutions, the found compound was MTX attached with fragment 3-aminopropylesilicic acid moiety (Scheme 1D and Fig. S1). There was no free MTX remaining in the MTX–PSi conjugation. Further quantification of the conjugation ratio was accessed by elemental analysis (Table S1) and calculated based on the increasing mass percentage of each element resulting from the conjugation related to the molecular Q5 formula of MTX and the surface chemistry of PSi. About 8.4 ± 1.6% (molar ratio) of the amine group in PSi was reacted with MTX, and thus, the mass loading of MTX to PSi was 5 lg/1 mg of nanoparticles.

The nanoparticle size and f-potential of both PSi and MTX–PSi were determined by DLS (Table 1 and Fig. 1B). The nanoparticles used in this work exhibited a nanosize distribution and free dispersibility in aqueous solutions with low polydispersity indices (PDI). The f-potential of MTX–PSi was slightly lower than that of bare PSi nanoparticles. The pKa-values of MTX are 4.8 and 5.5 [46], which present a slightly negative charge compared to the reacted amine groups on the surface of PSi at neutral conditions. Fig. 1C and D shows the morphologies of PSi and MTX–PSi nanoparticles obtained by TEM with an overview and closer observation (inside images). This result was consistent with our previous work on the PSi nanoparticle surface modification with peptides [17]. The porous structure of the MTX–PSi nanoparticles can be accessed for further drug loading.

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3.2. Cell uptake of MTX–PSi nanoparticles

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FR is widely over-expressed by a number of cancer cells. MTX has high affinity to FR, which can form FR-mediated cell uptake [24]. It has been demonstrated that MTX conjugated to dendrimers through amide linkage showed more than 4000-times higher affinity to FR than free MTX, and also multi-valent conjugate to one single dendrimer molecule showed a much higher affinity to FR than the free MTX to FR [31]. Thus, we hypothesize that MTX conjugated to PSi can increase the cellular uptake of the PSi nanoparticles in the high FR-expressing cells. On the other hand, the nanoparticle’s positive surface charge is beneficial for the non-specific cell internalization of the nanoparticles [47,48]. Cells with low FR-expression have very low cell uptake of MTX [49]. By conjugation of MTX to positively charged nanoparticles, the cell uptake of MTX could be increased [20]. Furthermore, the charge-based non-specific cell internalization can help to overcome the drug resistance of MTX. In order to evaluate the aforementioned capacity with our systems, PSi and MTX–PSi nanoparticles were incubated with the two cancer cells to determine the cell uptake efficacy. One cell line used was the hybrid endothelial EA.hy926 with a low expression of FR. The other cell line used was the human glioblastoma U87 MG, which highly over-expresses the FR. Fig. 2 shows qualitative TEM images of the PSi and MTX–PSi cell uptake by the EA.hy926 and U87 MG cells. MTX–PSi nanoparticles were uptaken by low FR

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Scheme 1. (A) MTX-conjugated to PSi by NHS/EDC coupling reaction to form MTX–PSi. (B) Molecular structure of MTX. (C) Released fragment from MTX–PSi. (D) Dehydrate form containing MTX determined by MS.



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Fig. 1. (A) ATR–FTIR of PSi and MTX–PSi. (B) Particle size distribution of PSi and MTX–PSi based on intensity values determined by DLS in Milli-Q water. (C and D) TEM pictures of PSi and MTX–PSi nanoparticles showing their characteristic morphology. Scale bars are 500 nm for the overview image and 200 nm for the insert images.

Table 1 Physiochemical characterization of the Z-average size, f-potential (in Milli-Q water) and drug loading degree of PSi and MTX–PSi. Data represent the mean ± s.d. of at least three independent measurements. The reported data are presented as the average of three independent measurements, i.e., the data are an average of three Z-average size values. The standard deviations were calculated based on the three Z-average size values. Sample

Z-average size (nm)

PDI

f-potential (mV)

Loading degree of sorafenib (w-%)

PSi MTX–PSi

177.0 ± 1.8 200.0 ± 1.7

0.106 ± 0.007 0.118 ± 0.05

+40.6 ± 1.5 +38.5 ± 1.9

6.1 ± 0.7 6.0 ± 0.6

Fig. 2. Intracellular uptake and distribution of the PSi and MTX–PSi nanoparticles in EA.hy926 and U87 MG cells. TEM images of ultrathin sections of EA.hy926 and U87 MG cells were used as controls or incubated with PSi and MTX–PSi nanoparticles at 37 °C (Inset scale bars 200 nm).

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expressed EA.hy926 cells, although there were less MTX–PSi nanoparticles located intracellularly than of the bare PSi nanoparticles. Amine-terminals on the surface of PSi enhance cellular uptake due to the positive charge interaction with the negative cell membrane and thus induce cellular endotheliosis [20]. The presence of MTX on the surface of PSi normalized the non-specific cell uptake of

MTX–PSi nanoparticles, and thus, led to a lower cell uptake. For the FR over-expressed U87 MG cells, there were more MTX–PSi nanoparticles located and distributed in the cytoplasm of the cells than with the bare PSi nanoparticles. This confirmed our hypothesis that MTX conjugation can increase the FR-mediated cell uptake by FR over-expressed cancer cells.



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MTX is a folic acid analog cancer drug which acts as an inhibitor of DHFR to stop the tetrahydrofolate synthesis and causes cell death [22]. The conjugation of MTX to PSi nanoparticles can increase the affinity to FR. However, DHFR inhibition using MTXdendrimer conjugates has been shown to be comparatively less active to DHFR than free MTX. This was presumably because only one MTX molecule on each dendrimer could bind to DHFR molecules [31]. From another report of MTX conjugated to carbon nanotubes (CNT), in vivo anticancer activities of the ester-linked MTX–CNT conjugates were significantly higher than the amidelinked counterpart [39]. This result suggested that the cleavability of linkers between MTX and the carrier was crucially determining the therapeutic performance of the conjugated MTX. Thus, our hypothesis is that the release of MTX from the PSi nanoparticles would be essential for increasing the efficacy of MTX in the developed cancer drug formulation. The aminosilane surface moiety can be hydrolyzed and detached from the PSi surface in an aqueous solution [19]. Thus the MTX fragment can be released from the PSi nanoparticles through hydrolysis. The released compound from the MTX–PSi was first examined by MS analysis. The main detected molecule by MS was MTX with a fragment of 3-aminopropylesilicic acid (Scheme 1D, Mw 555.2 g/mol; the compound was detected as the protonated molecule ([M+H]+, m/z 556.2)). The results confirmed that MTX with a fragment of (3-aminopropyl)trihydroxylsilane (Scheme 1C) was released from MTX–PSi, and then dehydrolyzed to the molecule presented in Scheme 1D. Next, the drug release profiles against time were analyzed by HPLC. Fig. 3A–C shows a sustained release of MTX from the MTX–PSi nanoparticles. MTX was released faster at pH 7.4 than at pH 5.5, due to the surface silane moiety that was hydrolyzed and detached from PSi faster at higher pH-values [19]. The MTX release profiles from MTX–PSi in buffer without FBS (Fig. 3B) and

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from MTX–PSi loaded with sorafenib in buffer with FBS (Fig. 3C) were similar to the release profiles of MTX–PSi in buffer with 10% FBS at both the pH-values (Fig. 3A). This was consistent with the results that the release of MTX was due to the cleavage of the aminosilane moiety from the PSi surface. This cleavage was not affected by serum proteins or by the loaded sorafenib in the PSi nanoparticles. During the first 12 h, there was approximately a linear sustained release of MTX in all the release conditions. Over 40% (pH 7.4) and 12% (pH 5.5) of MTX were released from the MTX–PSi nanoparticles during the first 12 h, followed by a sustained release up to 96 h, where the concentration of MTX reached 0.65 and 0.92 lg/mL at pH 5.5 and 7.4, respectively (Fig. 3B). In addition to the MTX conjugation to the PSi surface, another anti-angiogenic hydrophobic drug, sorafenib, was loaded into the MTX–PSi nanoparticles to explore potential combination therapy applications. After drug loading, 200 lg of each sorafenib loaded nanoparticle was resuspended in 1 mL of 48% mixture of ACN/ water solution and the supernatant was analyzed by HPLC. The concentrations were 12.9 and 12.7 lg/mL of sorafenib for PSi and MTX–PSi nanoparticles, respectively. The drug loading studies of sorafenib in PSi and MTX–PSi showed that the MTX conjugation to PSi did not affect the loading capacity of PSi nanoparticles (Table 1), leading to similar loading degrees of sorafenib both in the presence or absence of MTX. The dissolution rate of sorafenib in aqueous solution was, however, considerably enhanced by loading it into the both PSi nanoparticles (Fig. 4A and B). The solubility of sorafenib in 10% FBS aqueous buffer was 8.8 lg/mL. The dissolution studies were done in sink-conditions [17]. The concentration of sorafenib was 0.43 and 0.42 lg/mL after 8 h for PSi and MTX–PSi nanoparticles, respectively while only 0.03 lg/ mL of pure sorafenib was dissolved in the same condition at 8 h time point. These results were consistent with our previous work Q6 of peptides modification of PSi nanoparticles for drug delivery [17].

Fig. 3. Release profiles of MTX from MTX–PSi nanoparticles. (A) MTX release from MTX–PSi in buffer containing 10% FBS. (B) MTX release from MTX–PSi in buffer without FBS. (C) MTX release from sorafenib-loaded MTX–PSi nanoparticles in buffer containing 10% FBS.

Fig. 4. Dissolution profiles of sorafenib. (A) Sorafenib released from PSi and MTX–PSi nanoparticles in buffer (pH 7.4) containing 10% FBS, and (B) free sorafenib dissolution profile in buffer containing (pH 7.4) 10% FBS.



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Fig. 5. Cancer cell growth inhibition by treatment with pure MTX, PSi and MTX–PSi nanoparticles. The nanoparticles were suspended in full cell media. Cells were exposed to pure MTX, PSi and MTX–PSi nanoparticles for 24 (A: EA.hy926, C: U87 MG) and 48 h (B: EA.hy926, D: U87 MG) at 37 °C. Pure MTX was dissolved into DMSO (2 mg/mL). The cell viability of 0.1% of DMSO for EA.hy926 and U87 MG cells at 24 and 48 h is presented in Fig. S2.

Fig. 6. MTX–PSi nanoparticles inducing programed cell apoptosis. EA.hy926 and U87 MG cells were incubated with PSi and MTX–PSi nanoparticles for 24 h. After washing and harvesting, the cells were stained with 7-AAD for 15 min, followed by flow cytometry analysis to determine the percentage of DNA fragmentation in the samples. The MTX concentrations used were 0.1 and 0.5 lg/mL, which corresponded to the concentrations of 20 and 100 lg/mL for bare PSi and MTX–PSi nanoparticles.

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3.4. Inhibition of cancer cell proliferation

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In order to study the anti-proliferation efficacy of the MTX–PSi nanoparticles, EA.hy926 and U87 MG cells were exposed to free MTX or MTX–PSi with concentrations from 0.01 to 2 lg/mL for predetermined time, and the cells were quantified by an ATP-based cell proliferation assay (Fig. 5). Bare PSi nanoparticles at the same concentrations as the MTX–PSi nanoparticles were used as a control. At both 24 and 48 h incubation of MTX with EA.hy926, a minor effect on the cell proliferation was observed. Contrarily, MTX–PSi significantly inhibited the growth of EA.hy926 cells in a concentration-dependent manner, while the PSi nanoparticles had a modest effect on the cell growth (Fig. 5A and B). For the low folic acid receptor expressing EA.hy926 cells, free MTX had no effect on the cells up to concentrations of 2 lg/mL. However, after conjugation to the PSi nanoparticles, the half maximal inhibitory concentration (IC50) of MTX in EA.hy926 cells was 0.5 lg/mL at 24 h and 1.0 lg/mL at 48 h, respectively. This higher inhibition efficiency of MTX–PSi on the cell proliferation was probably due to the cationic surface of the nanoparticles enhancing the cellular uptake of MTX–PSi. After internalization, MTX was released from the MTX–PSi nanoparticles to the cytosol, inducing a higher cell death. Since free MTX had low cell uptake in EA.hy926 cells, its effect on cell proliferation was minimal.

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In the case of U87 MG cells, which highly express the FR, MTX can be internalized into the cells by FR-mediated cell uptake. The results showed that MTX successfully inhibited the U87 MG cell proliferation at both 24 and 48 h. MTX–PSi had a lower efficiency on the proliferation inhibition of U87 MG at concentrations lower than 0.5 lg/mL; however, at higher concentrations, MTX– PSi had comparable efficacy on the cell proliferation as free MTX, whereas the cell viability was close to the background levels (Fig. 5C and D). The IC50 of MTX in U87 cells was ca. 0.05 lg/mL at both 24 and 48 h incubation times. The IC50 of MTX–PSi was 0.5 lg/mL (24 h) and 0.25 lg/mL (48 h). The IC50 was increased when MTX was conjugated to the PSi nanoparticles. This might be due to the sustained release of MTX from MTX–PSi nanoparticles; there was a delay of MTX to inhibit the activity of DHFR enzyme.

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During the interphase of cell proliferation, the enzyme DHFR catalyzes the dihydrofolate to tetrahydrofolate for thymidylate synthesis intended for DNA duplication [50]. MTX can inhibit the activity of DHFR in the cytosol, resulting in inhibition of thymidylate synthesis and consequently stopping the DNA biosynthesis and leading to cell apoptosis [27]. To ensure that the cell death

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determined in cell proliferation experiments was due to the MTX-induced cell apoptosis, DNA fragmentation was accessed by 7-AAD stained and flow cytometry analysis. The percentage of the cells with DNA segmentation was measured for both EA.hy926 and U87 MG cells treated with PSi and MTX–PSi for 24 h (Fig. 6). In both cells, MTX–PSi caused a concentration-dependent DNA fragmentation. When treated with 0.1 lg/mL of MTX contained MTX–PSi nanoparticles, there was 25.7% and 11.2% of nuclear fragmentation for EA.hy926 and U87 MG cells, respectively. At a higher concentration (0.5 lg/mL), 80.8% and 72.8% for the EA.hy926 and U87 MG cells induced nuclear segmentation, respectively. Importantly, for both cell lines, the bare PSi nanoparticles did not cause a significant nuclear segmentation. These results demonstrated that MTX–PSi nanoparticles were up-taken by the cells and the biological activity of the released MTX was maintained.

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4. Conclusion

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PSi nanoparticles are versatile biomaterials with a nano-porous structure and large surface areas that can be used for drug loading and enhanced dissolution rate of poorly-water soluble drugs. MTX is a folate analog anticancer drug, which acts by inhibiting DHFR enzymatic activity in cytosol to induce cell death. In this work, MTX was successfully conjugated to amine-terminated PSi nanoparticles to obtain a non-specific cell uptake and to overcome potential drug resistance. The MTX conjugation did not affect the drug loading capacity of the PSi nanosystem. The hydrophobic anti-angiogenic drug, sorafenib, was successfully loaded to the MTX–PSi nanoparticles. The dissolution rate of this poorly-water soluble drug was dramatically enhanced due to the porous structure of the PSi nanosystem. Furthermore, MTX had a prolonged release from the MTX–PSi nanoparticles as a result of the covalent attachment to the surface of the PSi. The MTX release was due to the cleavage of 3-aminopropylsilane moiety from the surface of PSi nanoparticles. The cell viability showed that the MTX–PSi nanoparticles successfully inhibited the cell proliferation of both FR-over-expressed and low FR-expressing cells due to the induced MTX programed cell apoptosis. With this combination, fast released sorafenib can be expected to inhibit the neovascularization of the tumor in the tumor environment, while the improved cellular uptake of MTX–PSi nanoparticles and sustained release of chemotherapeutical MTX can inhibit the tumor cell proliferation for prolonged dosing times for cancer therapy.

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Conflict of interest

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The authors declare no competing financial interest. Acknowledgements C.-F. Wang acknowledges financial support from the Chinese Q7 Scholarship Council (Grant No. 2009627022). H.A. Santos acknowlQ8 edges the Academy of Finland (Decision nos. 252215 and 256394), the University of Helsinki Funds, Biocentrum Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013, Grant No. 310892) for financial support. We thank Dr. Anu Airaksinen and Dr. Mirkka Sarparanta (Department of Chemistry, University of Helsinki) for their kind help with the elemental analysis, and Dr. Pirjo Laakkonen (Research Programs Unit, Translational Cancer Biology and Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki,) for kindly provided the U87MG cells.

Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.01. 021.

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