Dual-Targeting Multifuntional Mesoporous Silica Nanocarrier for

Aug 3, 2017 - Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Env...
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Dual-Targeting Multifuntional Mesoporous Silica Nanocarrier for Codelivery of siRNA and Ursolic Acid to Folate Receptor Overexpressing Cancer Cells Guirong Zheng, Yiling Shen, Ruirui Zhao, Fan Chen, Ying Zhang, Aixiao Xu, and Jingwei Shao* Cancer Metastasis Alert and Prevention Center, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, and Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350116, China ABSTRACT: A targeting drug delivery system (TDDS) can selectively deliver antitumor drugs to cancerous parts to improve its anticancer efficacy. Hence, a targeted drug delivery system (UA/siVEGF@MSN-FA) coloading ursolic acid (UA) and vascular endothelial growth factor (VEGF) targeted siRNA (siVEGF) based on mesoporous silica (MSN) nanocarrier modified by a folic acid (FA) molecule was designed and synthesized. The MSN-FA nanoparticles were investigated for shape, diameter, and zeta potential and and by infrared (IR) spectroscopy. FR-overexpressing HeLa cells and FR-negative HepG2 cell lines were used to evaluate the in vitro cellular uptake and the cytotoxicity of MSN-FA nanoparticles. The morphology of HeLa cells transfected with siVEGF@MSN-FA was observed using fluorescence microscopy. Our findings demonstrated that UA@MSN-FA nanoparticles were near-spherical, and the particle size was about 209 ± 9.21 nm. The MSN-FA nanocarrier not only could enhance the in vitro transfection efficiency and the stability of siVEGF but also could further improve the targeted anticancer efficacy of UA and siVEGF via the active targeting property of FA. Overall, the MSN-FA drug delivery system could serve as an excellent material in biomedical applications. KEYWORDS: ursolic acid, siVEGF, mesoporous silica nanoparticles, folic acid, targeting drug delivery system targeting of the siRNA delivery system.18 A nanomaterial is a kind of typical nonviral vector, which has advantages of a small size and easy modification, and can effectively carry siRNA into cells and induce an RNAi effect.19 Therefore, finding a new nanodelivery system to improve the stability and targeting of the siRNA is extremely urgent. A targeting drug delivery system (TDDS), as a new drug delivery method, showed potential for the delivery of antitumor drug.20 Among various nanomaterials for drug delivery applications, mesoporous silica nanoparticles (MSNs) have attracted great attention due to their unique properties, including tunable pore size, prominent biocompatibility, significant chemical inertness, stable skeletal structure, good biocompatibility both in vitro and in vivo, and low cytotoxicity.21−25 MSNs have a huge specific surface area (>900 m2/g) and large pore volume (>0.9 cm3/g); it can improve the persistence of the drug efficacy.26−30 Moreover, MSNs can be easily developed as an efficient drug/gene codelivery system to achieve an expected therapeutic effect.31−33 In our previous work, a pH-sensitive prodrug delivery system (UA@MSN-UA) that incorporated an acidsensitive linkage between drug and silica-based mesoporous nanosphere (MSN) was successfully designed and synthesized. Also, this system can be a promising drug carrier for improving the bioavailability of UA.34 Very recently, we reported a tumor microenvironment-responsive controlled and sustained release

1. INTRODUCTION Ursolic acid (UA), 3β-hydroxy-urs-12-en-28-oic acid, is a pentacyclic triterpenoid that exists widely in food, medicinal herbs, and other plants.1−3 UA has many pharmacological properties, including anti-inflammatory,4 liver protective,5 antiatherosclerotic,6 antiepileptic,7 antidiabetic activities,8 and especially antitumor.9,10Though UA has great antitumor activity and low toxicity, the poor water solubility and low bioavailability restrict its further clinical application. To overcome this problem, more and more researchers have been working on structure modification of UA to obtain its highly active derivatives such as being modified to C-3, C12− C13 double bonds, or C-28.11 In our previous research, a number of UA derivatives (UP12, US597, Asp-UA)12−14 were successfully synthesized. Among them, US597 and Asp-UA could safely and effectively suppress cancer metastasis both in vitro and in vivo and is being developed as a novel cancer metastasis chemopreventive agent by us. However, the above applications for the improvement of UA characteristics are mainly focused on structure modification, most of the UA derivatives lack a tumor-targeting feature, and the synthesis procedure is relatively complicated. Thus, finding a new method to improve the bioavailability and solubility of UA as well as enhance its targeted anticancer efficacy is extremely urgent. Small interfering RNA as an effector of RNAi technology has been widely used in the field of gene therapy for various human diseases such as viral infections, genetic diseases, cardiovascular disorders, and cancers.15,16 However, siRNA as a drug for the treatment of disease; it still has many problems, mainly for the transmission stability of siRNA17 and high efficiency and © XXXX American Chemical Society

Received: July 3, 2017 Revised: July 20, 2017 Accepted: July 26, 2017

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DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Schematic illustration of the (A) synthesis procedure of UA/siVEGF@MSN-FA and (B) inhibiting effect on the proliferation of cancer cells. (APTES), ethylene glycol (EG), ammonia, ethanol, tetraethoxysilane (TEOS), and N,N-dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). 2.2. Synthesis of MSN-FA Nanoparticles. The MSN-NH2 was prepared as described previously.34 First, 2 g of CTAC, 0.1 g of TEA, and H2O (20 mL) were mixed; after stirring at 95 °C for 1 h, tetraethyl orthosilicate (TEOS, 1.5 mL) was added and then continuously stirred for 2 h. Then products were centrifuged at 13 000 rpm for 15 min and washed with ethanol and water. CTAC was extracted with 20 mL of methanol solution containing 0.2 g of NaCl. Then the products were centrifuged and washed with ethanol and water. After 100 mg of MSNOH was added to 20 mL of ethanol, then 400 μL of APTES was added. After the mixture solution was stirred for 24 h at room temperature, the mixture solution was centrifuged and washed three times with ethanol and water. The MSN-NH2 was dried with a vacuum dryer. The folic acid-modified MSN-NH2 (MSN-FA) was obtained. Briefly, 20 mg of FA was dissolved in 10 mL of DMF. After that 10 mg of EDC and 8 mg of NHS were added, and the mixture was stirred for 12 h at room temperature. Then 30 mg of MSN-NH2 was added to the mixture and stirred overnight at room temperature; the products were collected by centrifugation and washed with pure ethanol and deionized water. Then we centrifuged the solution and dried the final products with a vacuum dryer at −50 °C for 24 h. 2.3. Preparation of FITC-Labeled MSN-FA (FMSN-FA). First, 2 mg of FITC was dissolved in 2 mL of ethanol, and then 20 μL of APTES was added to the solution and stirred 24 h in the dark. Then, 100 mg of MSN-FA was added in 20 mL of ethanol and mixed with 2 mL of FITC/APTES ethanol solution and then stirring for 24 h under dark conditions. The products (FMSN-FA) were centrifuged and washed three times with pure ethanol. Finally, the products (FMSNFA) should be dried with a vacuum dryer at −50 °C for 24 h. 2.4. Characterization of MSN, MSN-FA, and FMSN-FA. The side distribution and zeta potential of MSN, MSN-FA, and FMSN-FA were measured by Zetasizer NanoZS90 (Malver, USA). The morphology of MSN was characterized by a transmission electron microscope (TEM, Japan). Nitrogen adsorption−desorption analysis was measured on an adsorption analyzer (V-Sorb 2800P, Beijing,

drug delivery system based on MSN-CS-FA nanocarrier for targeting delivery of UA to cancer cells; it could help broaden the usage of UA and reflect the great application potential of the UA as an anticancer agent.35 Thus far, however, there are no reports about using MSN as both UA and siRNA codelivery system. In the present study, a mesoporous silica (MSN) nanocarrier modified by a folic acid (FA) molecule was designed and synthesized (Figure 1). Folic acid (FA) was first covalently conjugated to the surface of MSN through an acid-labile amide bond. Afterward, UA was encapsulated inside the MSN-FA by noncovalent interactions. Finally, siVEGF were loaded into the nanocarrier by electrostatic interaction. We hypothesized that MSN-FA nanocarrier coloaded with UA and siVEGF could lead to increasing solubility and enhancing bioavailability of UA, as well as improving the stability of siVEGF. The targeting of the MSN-FA nanocarrier allows UA and siVEGF to be enriched in the tumor site and plays a synergistic antitumor effect. The shape, diameter, zeta potential, encapsulation efficiency, and drug loading and infrared (IR) spectroscopy of nanoparticles were investigated. Then we conducted the drug release behavior of free UA and UA@MSN-FA. Furthermore, the UA/siVEGF@MSN-FA prodrug nanoparticle was subjected to biological evaluation against HeLa and HepG2 cells by cellular uptake, CCK8 study, in vitro transfection, and Western blot assay.

2. MATERIALS AND METHODS 2.1. Chemicals. Hexadecyl trimethylammonium chloride (CTAC), triethanolamine (TEA), and folic acid (FA) were purchased from Aladdin Reagents Co., Ltd. (Shanghai,China). Ursolic acid (UA) was obtained from BSISL (Hunan, China). Green fluorescent protein (GFP)-labeled siVEGF was synthesized by Sangon Biotech (Shanghai, China). Cell Counting Kit-8 was purchased from Beyotime Biotechnology (Shanghai, China). 3-Aminopropyltriethoxysilane B

DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Characterization of the nanoparticle. (A) DLS analysis of MSN; (B) DLS analysis of MSN-FA; (C) zeta potential analysis of MSN-OH, MSN-NH2, MSN-F,A and FMSN-FA; (D) TEM image of MSN; BET nitrogen adsorption−desorption isotherms (E) and BJH pore size distributions (F) of MSN. 2.7. In Vitro Drug Release of Nanoparticles. To determine the in vitro release of UA@MSN and UA@MSN-FA, nanoparticles were dispersed in PBS (pH 7.4) and obtained a final concentration of 1 mg/ mL. Then 2 mL of the solution was loaded in a dialysis bag and put in PBS (pH 7.4). Subsequently, the release characteristic of nanoparticles was detected by constant temperature vibration dialysis assay at 37 °C. With a certain interval off time to sample, the in vitro drug release rates were detected by quantitative analysis by UV−vis. Finally, the in vitro drug release rates line with time was drawn in the diagram. 2.8. Cellular Uptake. Fluorescein-labeled MSN and MSN-FA were fabricated as described above. HeLa and HepG2 cells were seeded in a 24-well plate at a density of 1 × 105 per well and incubated overnight. After that the cells were treated with 100 μg/mL FITC, FITC@MSN, and FITC@MSN-FA for 2 h at 37 °C; the cells were washed three times with cold PBS to remove the residue. Also, the nuclei were stained with Hoechst 33342 for 10 min, and then the cells

China). Fourier transform infrared spectrophotometric (FT-IR) spectra of FA, MSN-NH2, MSN-FA and FMSN-FA were measured on a FT-IR spectrometer (Bruker IFS 55, Fällanden, Switzerland) to scan over a region from 400 to 4000 cm−1 on a thin KBr slice. 2.5. Preparation of UA-Loaded MSN-FA. Briefly, 30 mg of MSN/MSN-FA was dispersed into 30 mL of acetone solution and ultrasonically dissolving for 1 h. Then 20 mg of UA was added to the solution and stirred 24 h at room temperature. After centrifugation, the supernatant was retained. Finally, the residue was washed with deionized water and products dried with a vacuum dryer to get the final products. 2.6. Preparation of siVEGF-Loaded UA@MSN-FA. Briefly, 20 mg of UA@MSN-FA was dispersed into 20 mL of acetone solution and ultrasonically dissolving for 1 h. Then 6 μL of UA@MSN-FA and 2 μg of siVEGF were stirred gently for 20 min to form the complexes of UA/siVEGF@MSN-FA nanoparticles. C

DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Characteristics of MSN-OH, FMSN-FA, UA@MSN-NH2, and UA@MSN-FA nanoparticles MSN-OH FMSN-FA UA@MSN-NH2 UA@MSN-FA

diameter (nm) 94.3 243 158 209

± ± ± ±

7.40 10.5 5.93 9.21

PDI 0.26 0.31 0.26 0.23

zeta potential (mV)

±0.02 ± 0.10 ± 0.03 ± 0.07

−17.9 −13.6 25.3 −12.2

were washed three times with PBS. Last, the cells were measured using a Leica confocal microscope (SP-8, Germany). 2.9. In Vitro Cell Viability Study. Mesoporous silica nanoparticles can stimulate exocytosis of MTT formazan crystals.36 Thus, we used CCK8 to detect the cytotoxicity of UA/siVEGF@MSN-FA on HepG2 and HeLa cells.37 Cells were seeded in a 96-well plate at a density of 7000 cells per well and incubated for 24 h at 37 °C with 5% CO2. Then MSN-NH2, MSN-FA, FMSN-FA, free UA, UA@MSN, or UA/siVEGF@MSN-FA (siVEGF = 2 μg) was added to the cells in DMEM at different concentrations and incubated at 37 °C with 5% CO2 for 24 h. Then 10 μL of CCK8 (VP757, Dojindo Laboratories, Kumamoto, Japan) was supplemented onto each well and then cultured for 2 h. Absorbance was detected on a microplate ELISA reader (Tecan, Switzerland) at 450 nm. 2.10. In Vitro Transfection. In vitro transfection efficiency of siVEGF@MSN-FA in HeLa cells was examined by fluorescence microscopy. A 6 μL amount of MSN or MSN-FA and 2 μg of siVEGFGFP were stirred gently for 20 min to form the complexes of siVEGF@MSN or siVEGF@MSN-FA. HeLa cells (5 × 105 cells per well) were plated in 24-well plates for 24 h. Also, cells were incubated with free siVEGF, siVEGF@MSN, and siVEGF@MSN-FA at 37 °C and transfected for 48 h. After the transfection is complete, the cells were observed with an Olympus IX71 fluorescence microscope (Melville, NY, USA). 2.11. Serum Stability Assay. Serum stability assay was performed as described previously.38 siVEGF@MSN-FA were prepared as described above; 3 μL of free siRNA (siVEGF = 1 μg) and 6 μL of siVEGF@MSN-FA (MSN-FA, 3 μL; siRNA, 3 μL; siVEGF = 1 μg) were incubated with the same volume of fresh mouse serum and then 37 °C incubation at different time points (0, 2, 4, 6, 8, 12, and 24 h). Electrophoresis was carried out on 2% agarose gel with a current of 80 V for 30 min in TAE buffer solution (40 mM Tris-HCl, 1 v/v % acetic acid, and 1 mM EDTA). The agarose gel detection was revealed by using ImageJ. 2.12. Western Blot Assay. Western blot assay was performed as described previously.39,40 HeLa cells (5 × 105 cells per well) were incubated in 6-well plates for 24 h. After that the cells were treated with free siVEGF, siVEGF@MSN, and siVEGF@MSN-FA (siVEGF = 2 μg/well) for 24 h. Cells were suspended in ice-cold lysis buffer and lysed for several seconds, and then the supernatant upper sample was clarified by centrifugation at 12 000 g for 10 min under 4 °C. Protein concentrations were determined by bicinchoninic acid (BCA). The denatured samples (20 μL purified protein) were resolved on 10% SDS-PAGE gels. Afterward, proteins were transferred to polyvinylidene (PVDF). The PVDF was washed for 10 min with TBS and blocked with tris-buffered saline (TBST) which contained 5% (w/v) nonfat dry milk for over 2 h. Next, the PVDF was washed three times with TBST and incubated with a proper dilution of specific primary antibodies in TBST overnight at 4 °C. After washing with TBST, the PVDF was incubated with a suitable secondary antibody (horseradish proxidase-conjugated goat antimouse or antirabbit lgG) for 2 h at 4 °C. Subsequently, the particular protein was visualized with the ECL kit. Band detection was revealed by using ImageJ. 2.13. Statistical Analysis. Data were expressed as the mean ± standard deviation (SD) of triplicates. Differences were considered statistically significant at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). All statistical treatments were performed using the SPSS 17.0 software.

± ± ± ±

0.54 1.26 1.83 1.35

encapsulation efficiency (%)

drug loading (%)

57.26 ± 1.97 49.1 ± 1.52

18.9 ± 2.03 18.5 ± 2.84

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of MSN and MSN-FA. The synthetic procedures of UA/siVEGF@MSN-FA

Figure 3. Characterization of the nanoparticle. (A) Fourier-transform IR spectra of MSN, FA, MSN-FA, and FMSN-FA; (B) in vitro release of UA@MSN-FA. Free UA release rate is compared with UA@MSNFA in PBS (pH = 7.4) at 37 °C.

are shown in Figure 1A. First, MSN nanoparticles were synthesized and the size was optimized, and CTAC was removed from the pore of nanoparticles by anhydrous ethanol and concentrated hydrochloric acid. Second, the surface of nanoparticles was modified with an amino group by reacting with APTES, and FA was conjugated to the surfaces of MSNNH2 by amino and carboxyl condensation. Then UA was encapsulated inside the MSN-FA by noncovalent interactions. Afterward, siVEGF was loaded into the nanocarrier by electrostatic interaction. Finally, UA/siVEGF@MSN-FA actively entered into the cancer cell, and UA and siVEGF effectively escaped from MSNs nanoparticles (Figure 1B). The size and features of the MSN were characterized by TEM images and N2 adsorption−desorption isotherms. As shown in Figure 2D, the size of MSN was approximately D

DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Cellular uptake of MSN-FA. Confocal laser scanning microscopy images of MSN-FA taken up by HeLa and HepG2 cells. HeLa and HepG2 cells were incubated with FITC-MSN, FITC-MSN-FA, or without any treatment as a control for 2 h at 37 °C. Then cells were incubated with Hoechst 33342 for 10 min at 37 °C.

spherical. The side distribution of MSN was restricted, and the average diameter of it was about 130 nm. The physical properties of MSN were analyzed by N2 adsorption− desorption isotherms (Figure 2E and 2F), and the surface area (1056.7 m2/g), pore volume (1.14 cm3/g), and BJH pore size distribution (2.7 nm) were obtained. This illustrates the products of MSN have better uniformity and dispersity. The size diameter of nanoparticles was measured by dynamic light scattering (DLS). As shown in Figure 2A, the average diameter of MSN was about 160 nm and bigger than the result of TEM. Because TEM measures the size of dry nanoparticles and Malvern is a measure of the size of hydrated nanoparticles, the surface of nanoparticles became a hydrolysis layer so the size was enlarged. As seen in Figure 2B, the average diameter of MSN-FA was about 200 nm.

The zeta potential of MSN-OH, MSN-NH2, MSN-FA, and FMSN-FA was characterized at the pH 7.4 by a Malvern laser particle size analyzer (Malver, USA). As shown in Figure 2C, the zeta potential of MSN-OH or MSN-NH2 or FMSN-FA or MSN-FA was −17.9 ± 3.5, 25.34 ± 1.8, −13.6 ± 1.2, and −12.2 ± 1.3 mV, respectively. The results confirmed that the synthesis of FMSN-FA was successful. The drug loading efficiencies of MSN-NH2 and MSN-FA were all around 18%. However, the encapsulation efficiency of MSN-NH2 or MSN-FA was 57.6 ± 1.9% or 49.1 ± 1.5% (Table 1), respectively. Fourier transform infrared spectrophotometric (FT-IR) spectra of FA, MSN-NH2, FMSN-FA, and MSN-FA were measured on a FT-IR spectrophotometer. The FT-IR spectra of MSN-NH2 are shown in Figure 3A; the spectra of samples displayed peaks at 1050 and 1539 cm−1, which were a Si−O−Si E

DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Effects of UA@MSN-FA on the cell viability of HeLa and HepG2 cells. (A) HeLa and HepG2 cells were incubated with different concentrations of free UA, UA@MSN, or UA/siVEGF@MSN-FA (siVEGF = 2 μg) for 24 h. (B) HeLa and HepG2 cells were incubated with different concentrations of MSN-NH2, MSN-FA, and FMSN-FA for 24 h.

through CCK8 assay. As shown in Figure 5B, when HepG2 and HeLa cells were treated with MSN-NH2, MSN-FA, and FMSN-FA, these cancer cells still survived well, indicating that these nanoparticles were biocompatible and produced no significant toxicity to tissues and cells. Compared with free UA, UA@FMSN, and UA@FMSN-FA, UA/siVEGF@FMSN-FA was more cytotoxic to HepG2 and HeLa cells (Figure 5A). However, compared with FR negative HepG2 cell, the targeting drug delivery system FMSN-FA could effectively improve the antitumor effect of UA and siVEGF in FR overexpressing HeLa cells. 3.5. In Vitro Transfection. The siRNA transfection activities of siVEGF@MSN-FA were investigated using HeLa cells. As shown in Figure 6A and 6B; the transfection efficiency of free siVEGF was 4.5%, which was lower than siVEGF@MSN (23%), showing that the nanoparticles can increase the transfection efficiency. siVEGF@MSN-FA could enhance the siVEGF transfection activity compared to others remarkably, presenting a transfection efficiency of 45%. The result comfirmed that FA-modifided nanoparticles could improve the transfection efficiency extremely. 3.6. Stability of siVEGF@MSN-FA in Serum. Free siRNA can be degraded by endonucleases in human plasma with a very short half-life.41 Whether the MSN-FA nanoparticles could improve the stability of siRNA in serum was determined by gel retardation assay. As shown in Figure 6C, free siVEGF has been substantially completely degraded at 4 h while siVEGF in MSN-FA nanoparticles still exists at 6 h. It demonstrated that MSN-FA could effectively improve the stability of siVEGF in serum and play a role in the effect of treatment cancer. 3.7. Effect of siVEGF@MSN-FA on VEGF Protein. Western blot assay further invstigated the transfection of siVEGF@MSN-FA to HeLa cells. As shown in Figure 7A and 7B, free siVEGF could not reduce VEGF expression, and there is little difference with control. siVEGF@MSN could inhibit the expression of VEGF more than free siVEGF or control. In addition, the expressive level of VEGF was degraded

stretching vibration and an amino group vibration, respectively. The surface grafting of MSN-FA onto the MSN-NH2 surface was validated by two new absorbance bands at 1698 cm−1 of the stretching vibration of the CO groups and 1560 cm−1 of the amide band, indicating that the carboxyl groups had been introduced onto the MSN nanoparticles via the amide linkage. After coating the FITC, several new absorption signals appeared. The peak appearing at 1600−1400 cm−1 was attributed to the aromatic ring skeleton stretching vibration, and the absorbance located at 1635 cm−1 was assigned to the stretching vibrations of N−H, which supported the FITC coating on the surface of MSN-FA. These results confirmed that the grating process is successful. 3.2. In Vitro Drug Release of UA@MSN-FA and UA. To confirm whether the UA@MSN-FA nanoparticles could be an effective drug delivery system for sustained release of UA, we performed the drug release experiment under pH 7.4 for 72 h. As shown in Figure 3B, it was observed that 80% of free UA was released within 10 h, whereas UA-loaded NP (UA@MSNFA) showed a sustained release profile in release media. The in vitro drug release rate of UA@MSN-FA was maintained at 75% after 48 h. It demonstrated that UA@MSN-FA is more able to release UA and exhibited sustained drug release. 3.3. Intracellular Uptake of MSN-FA. FR-overexpressing HeLa cells and FR-negative HepG2 cells were used to study the cellular uptake of MSN-FA. As shown in Figure 4, compared with the green fluorescent intensities of free FITC and FITC@ MSN, no matter in HeLa or HepG2 cells, the green fluorescent intensities of FITC@MSN-FA are the strongest. Furthermore, the green fluorescent intensities of MSN-FA were much stronger in HeLa cell, indicating that MSN-FA could effectively target FR overexpressing HeLa cells. 3.4. Cytotoxicity Study. HepG2 and HeLa cells were used to study the cytotoxicity of UA/siVEGF@MSN-FA. To determine the anticancer ability in vitro of UA/siVEGF@ MSN-FA, we compared it with the cytotoxicity of MSN-NH2, MSN-FA, FMSN-FA, UA, UA@MSN, and UA@MSN-FA F

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Figure 7. Effects of siVEGF@MSN-FA NPs on the expression levels of VEGF protein. Expressions of VEGF in HeLa cells under control, siVEGF, siVEGF@MSN, and siVEGF@MSN-FA were detected by Western blotting.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Phone: + 86-13600802402. Fax: +86-591-22867183. ORCID

Jingwei Shao: 0000-0002-2060-8887 Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This project was supported by the National Science Foundation of China (81472767, 81673698, and 81201709).

Figure 6. In vitro transfection and the stability of siVEGF@MSN-FA. (A and B) Fluorescence field images of HeLa cells transfected for free siVEGF, siVEGF@MSN, and siVEGF@MSN-FA. (C) Stability of free siRNA and siVEGF@MSN-FA in serum.

REFERENCES

(1) Price, K. R.; Johnson, I. T.; Fenwick, G. R. The chemistry and biological significance of saponins in foods and feedingstuffs. Crit Rev. Food Sci. Nutr 1987, 26, 27−135. (2) Mahato, S. B.; Garai, S. Triterpenoid saponins. Fortschr Chem. Org. Naturst 1998, 74, 1−196. (3) Han, D. W.; Ma, X. H.; Zhao, Y. C.; Yin, L.; Ji, C. X. Studies on the preventive action of oleanolic acid on experimental cirrhosis. J. Tradit. Chin. Med. 1982, 2, 83−90. (4) Harmand, P. O.; Duval, R.; Delage, C.; Simon, A. Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells. Int. J. Cancer 2005, 114, 1−11. (5) Song, M.; Hang, T. J.; Wang, Y.; Jiang, L.; Wu, X. L.; Zhang, Z.; Shen, J.; Zhang, Y. Determination of oleanolic acid in human plasma and study of its pharmacokinetics in Chinese healthy male volunteers by HPLC tandem mass spectrometry. J. Pharm. Biomed. Anal. 2006, 40, 190−6. (6) Balanehru, S.; Nagarajan, B. Protective effect of oleanolic acid and ursolic acid against lipid peroxidation. Biochem. Int. 1991, 24, 981−90. (7) Liu, J. Pharmacology of oleanolic acid and ursolic acid. J. Ethnopharmacol. 1995, 49, 57−68. (8) Zhong, T.; Yao, X.; Zhang, S.; Guo, Y.; Duan, X.-C.; Ren, W.; Huang, D.; Yin, Y.-F.; Zhang, X., A self-assembling nanomedicine of conjugated linoleic acid-paclitaxel conjugate (CLA-PTX) with higher

dramatically when siVEGF was loaded in MSN-FA. This result indicated that siVEGF@MSN-FA could effectively improve the transfection efficiency of siVEGF and inhibit the expression of VEGF. In summary, a FA-conjugated mesoporous silica nanocarrier system was designed and synthesized. The MSN-FA has a uniform size of about 180 nm. The in vitro release results indicated that UA@MSN-FA exhibited a sustained release profile in the initial 20 h. For in vitro cellular uptake assay, we found that FA could improve the targeting property of the drug delivery system greatly. Compared with free UA, this targeted codrug delivery system (UA/siRNA@MSN-FA) could effectively enhance cell cytotoxicity effects on FR overexpressing cancer cells. Furthermore, in vitro transfection and Western blot assay results showed that MSN-FA could effectively improve the transfection effcicency of siRNA and significant inhibition the expression of cancer-related VEGF proteins in HeLa cells. Therefore, MSN-FA could be a promising nanocarrier of antitumor drugs for cancer treatment and a targeted drug delivery system to enhance antitumor efficiency. G

DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry drug loading and carrier-free characteristic. Sci. Rep. 2016, 6.10.1038/ srep36614 (9) Prasad, S.; Yadav, V. R.; Kannappan, R.; Aggarwal, B. B. Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-induced apoptosis through p53-independent up-regulation of death receptors: evidence for the role of reactive oxygen species and JNK. J. Biol. Chem. 2011, 286, 5546−57. (10) Shanmugam, M. K.; Rajendran, P.; Li, F.; Nema, T.; Vali, S.; Abbasi, T.; Kapoor, S.; Sharma, A.; Kumar, A. P.; Ho, P. C.; Hui, K. M.; Sethi, G. Ursolic acid inhibits multiple cell survival pathways leading to suppression of growth of prostate cancer xenograft in nude mice. J. Mol. Med. (Heidelberg, Ger.) 2011, 89, 713−27. (11) Sha, Y.; Yan, M. C.; Liu, J.; Liu, Y.; Cheng, M. S. Facile synthesis of oleanolic acid monoglycosides and diglycosides. Molecules 2008, 13, 1472−86. (12) Dong, H.; Yang, X.; Xie, J.; Xiang, L.; Li, Y.; Ou, M.; Chi, T.; Liu, Z.; Yu, S.; Gao, Y.; Chen, J.; Shao, J.; Jia, L. UP12, a novel ursolic acid derivative with potential for targeting multiple signaling pathways in hepatocellular carcinoma. Biochem. Pharmacol. 2015, 93, 151−62. (13) Xiang, L.; Chi, T.; Tang, Q.; Yang, X.; Ou, M.; Chen, X.; Yu, X.; Chen, J.; Ho, R. J.; Shao, J.; Jia, L. A pentacyclic triterpene natural product, ursolic acid and its prodrug US597 inhibit targets within cell adhesion pathway and prevent cancer metastasis. Oncotarget 2015, 6, 9295−312. (14) Tang, Q.; Liu, Y.; Li, T.; Yang, X.; Zheng, G.; Chen, H.; Jia, L.; Shao, J. A novel co-drug of aspirin and ursolic acid interrupts adhesion, invasion and migration of cancer cells to vascular endothelium via regulating EMT and EGFR-mediated signaling pathways: multiple targets for cancer metastasis prevention and treatment. Oncotarget 2016, 7, 73114−73129. (15) Lares, M. R.; Rossi, J. J.; Ouellet, D. L. RNAi and small interfering RNAs in human disease therapeutic applications. Trends Biotechnol. 2010, 28, 570−9. (16) Kim, D. H.; Rossi, J. J. Strategies for silencing human disease using RNA interference. Nat. Rev. Genet. 2007, 8, 173−84. (17) Choi, K. M.; Choi, S. H.; Jeon, H.; Kim, I. S.; Ahn, H. J. Chimeric capsid protein as a nanocarrier for siRNA delivery: stability and cellular uptake of encapsulated siRNA. ACS Nano 2011, 5, 8690− 9. (18) Torchilin, V. P. Multifunctional, stimuli-sensitive nanoparticulate systems for drug delivery. Nat. Rev. Drug Discovery 2014, 13, 813−27. (19) Khan, O. F.; Zaia, E. W.; Jhunjhunwala, S.; Xue, W.; Cai, W.; Yun, D. S.; Barnes, C. M.; Dahlman, J. E.; Dong, Y.; Pelet, J. M.; Webber, M. J.; Tsosie, J. K.; Jacks, T. E.; Langer, R.; Anderson, D. G. Dendrimer-Inspired Nanomaterials for the in Vivo Delivery of siRNA to Lung Vasculature. Nano Lett. 2015, 15, 3008−16. (20) Jain, V.; Jain, S.; Mahajan, S. C. Nanomedicines based drug delivery systems for anti-cancer targeting and treatment. Curr. Drug Delivery 2015, 12, 177−91. (21) Shang, F.; Sun, J.; Wu, S.; Liu, H.; Guan, J.; Kan, Q. Direct synthesis of acid-base bifunctionalized hexagonal mesoporous silica and its catalytic activity in cascade reactions. J. Colloid Interface Sci. 2011, 355, 190−7. (22) Liu, T.; Li, L.; Teng, X.; Huang, X.; Liu, H.; Chen, D.; Ren, J.; He, J.; Tang, F. Single and repeated dose toxicity of mesoporous hollow silica nanoparticles in intravenously exposed mice. Biomaterials 2011, 32, 1657−68. (23) He, Q.; Gao, Y.; Zhang, L.; Zhang, Z.; Gao, F.; Ji, X.; Li, Y.; Shi, J. A pH-responsive mesoporous silica nanoparticles-based multi-drug delivery system for overcoming multi-drug resistance. Biomaterials 2011, 32, 7711−20. (24) Li, L.; Tang, F.; Liu, H.; Liu, T.; Hao, N.; Chen, D.; Teng, X.; He, J. In vivo delivery of silica nanorattle encapsulated docetaxel for liver cancer therapy with low toxicity and high efficacy. ACS Nano 2010, 4, 6874−82. (25) Lu, J.; Liong, M.; Zink, J. I.; Tamanoi, F. Mesoporous silica nanoparticles as a delivery system for hydrophobic anticancer drugs. Small 2007, 3, 1341−6.

(26) Chen, C.; Geng, J.; Pu, F.; Yang, X.; Ren, J.; Qu, X. Polyvalent nucleic acid/mesoporous silica nanoparticle conjugates: dual stimuliresponsive vehicles for intracellular drug delivery. Angew. Chem., Int. Ed. 2011, 50, 882−6. (27) Fan, W.; Shen, B.; Bu, W.; Chen, F.; He, Q.; Zhao, K.; Zhang, S.; Zhou, L.; Peng, W.; Xiao, Q.; Ni, D.; Liu, J.; Shi, J. A smart upconversion-based mesoporous silica nanotheranostic system for synergetic chemo-/radio-/photodynamic therapy and simultaneous MR/UCL imaging. Biomaterials 2014, 35, 8992−9002. (28) Fu, C.; Liu, T.; Li, L.; Liu, H.; Chen, D.; Tang, F. The absorption, distribution, excretion and toxicity of mesoporous silica nanoparticles in mice following different exposure routes. Biomaterials 2013, 34, 2565−75. (29) Zhao, Y. N.; Vivero-Escoto, J. L.; Slowing, I. I.; Trewyn, B. C.; Lin, V. S. Y. Capped mesoporous silica nanoparticles as stimuliresponsive controlled release systems for intracellular drug/gene delivery. Expert Opin. Drug Delivery 2010, 7, 1013−1029. (30) Thomas, M. J.; Slipper, I.; Walunj, A.; Jain, A.; Favretto, M. E.; Kallinteri, P.; Douroumis, D. Inclusion of poorly soluble drugs in highly ordered mesoporous silica nanoparticles. Int. J. Pharm. 2010, 387, 272−7. (31) Shen, J.; Kim, H. C.; Su, H.; Wang, F.; Wolfram, J.; Kirui, D.; Mai, J.; Mu, C.; Ji, L. N.; Mao, Z. W.; Shen, H. Cyclodextrin and polyethylenimine functionalized mesoporous silica nanoparticles for delivery of siRNA cancer therapeutics. Theranostics 2014, 4, 487−97. (32) Ma, X.; Zhao, Y.; Ng, K. W.; Zhao, Y. Integrated hollow mesoporous silica nanoparticles for target drug/siRNA co-delivery. Chem. - Eur. J. 2013, 19, 15593−603. (33) Powell, C. J.; Werner, W. S.; Shard, A. G.; Castner, D. G. Evaluation of Two Methods for Determining Shell Thicknesses of Core-Shell Nanoparticles by X-ray Photoelectron Spectroscopy. J. Phys. Chem. C 2016, 120, 22730−22738. (34) Li, T.; Chen, X.; Liu, Y.; Fan, L.; Lin, L.; Xu, Y.; Chen, S.; Shao, J. pH-Sensitive mesoporous silica nanoparticles anticancer prodrugs for sustained release of ursolic acid and the enhanced anti-cancer efficacy for hepatocellular carcinoma cancer. Eur. J. Pharm. Sci. 2017, 96, 456−463. (35) Jiang, K.; Chi, T.; Li, T.; Zheng, G.; Fan, L.; Liu, Y.; Chen, X.; Chen, S.; Jia, L.; Shao, J. A smart pH-responsive nano-carrier as a drug delivery system for the targeted delivery of ursolic acid: suppresses cancer growth and metastasis by modulating P53/MMP-9/PTEN/ CD44 mediated multiple signaling pathways. Nanoscale 2017, 9, 9428−9439. (36) Fisichella, M.; Dabboue, H.; Bhattacharyya, S.; Saboungi, M. L.; Salvetat, J. P.; Hevor, T.; Guerin, M. Mesoporous silica nanoparticles enhance MTT formazan exocytosis in HeLa cells and astrocytes. Toxicol. In Vitro 2009, 23, 697−703. (37) Lin, X.; Zheng, L.; Song, H.; Xiao, J.; Pan, B.; Chen, H.; Jin, X.; Yu, H. Effects of microRNA-183 on epithelial-mesenchymal transition, proliferation, migration, invasion and apoptosis in human pancreatic cancer SW1900 cells by targeting MTA1. Exp. Mol. Pathol. 2017, 102, 522−532. (38) Oh, B.; Lee, M. Combined delivery of HMGB-1 box A peptide and S1PLyase siRNA in animal models of acute lung injury. J. Controlled Release 2014, 175, 25−35. (39) Zheng, G. R.; Shen, Z. C.; Chen, H. N.; Liu, J.; Jiang, K.; Fan, L. L.; Jia, L.; Shao, J. W. Metapristone suppresses non-small cell lung cancer proliferation and metastasis via modulating RAS/RAF/MEK/ MAPK signaling pathway. Biomed. Pharmacother. 2017, 90, 437−445. (40) Shao, J.; Zheng, G.; Chen, H.; Liu, J.; Xu, A.; Chen, F.; Li, T.; Lu, Y.; Xu, J.; Zheng, N.; Jia, L., Metapristone (RU486 metabolite) suppresses NSCLC by targeting EGFR-mediated PI3K/AKT pathway. Oncotarget 2017.10.18632/oncotarget.18640 (41) Prabha, S.; Vyas, R.; Gupta, N.; Ahmed, B.; Chandra, R.; Nimesh, S. RNA interference technology with emphasis on delivery vehicles-prospects and limitations. Artif. Cells, Nanomed., Biotechnol. 2016, 44, 1391−1399.

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DOI: 10.1021/acs.jafc.7b03047 J. Agric. Food Chem. XXXX, XXX, XXX−XXX