Biotin-Modified Polylactic-co-Glycolic Acid Nanoparticles with

Article Cite This: J. Agric. Food Chem. 2018, 66, 9219−9230

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Biotin-Modified Polylactic-co-Glycolic Acid Nanoparticles with Improved Antiproliferative Activity of 15,16-Dihydrotanshinone I in Human Cervical Cancer Cells Jingjing Luo,†,‡,○ Xiaofeng Meng,†,‡,○ Jianyu Su,*,†,‡,§ Hang Ma,∥ Wen Wang,†,‡ Liming Fang,⊥ Huade Zheng,⊥ Yexia Qin,# and Tianfeng Chen*,∇ †

School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, Guangzhou 510640, China § Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111 Center), Guangzhou 510640, China ∥ Bioactive Botanical Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, United States ⊥ Department of Materials Science and Engineering, South China University of Technology, Guangzhou 510640, China # Institute of Industrial Technology Research, South China University of Technology, Guangzhou 510640, China ∇ Department of Chemistry, Jinan University, Guangzhou, 510632, China

J. Agric. Food Chem. 2018.66:9219-9230. Downloaded from pubs.acs.org by UNIV OF FINDLAY on 09/11/18. For personal use only.

‡

ABSTRACT: 15,16-Dihydrotanshinone I (DI), a natural compound isolated from a traditional Asian functional food Salvia Miltiorrhiza Bunge, is known for its anticancer activity. However, poor solubility of DI limits its desirable anticancer application. Herein, polylactic-co-glycolic acid (PLGA) was functionalized with polyethylene glycol (PEG) and biotin to form copolymers PEG-PLGA (PPA) and biotin-PEG-PLGA (BPA). DI was encapsulated in copolymers PPA and BPA to obtain DI-PPA-NPs (NPs = nanoparticles) and DI-BPA-NPs, respectively. The particle size and its distribution, encapsulation efficiency, and in vitro releasing capacity of DI-BPA-NPs were characterized by biophysical methods. MTT assay was used to evaluate the antiproliferative activity of free DI, DI-PPA-NPs, and DI-BPA-NPs in human cervical cancer Hela cells. DI-BPA-NPs showed the highest cytotoxicity on Hela cells with an IC50 value of 4.55 ± 0.631 μM, while it was 8.20 ± 0.849 and 6.14 ± 0.312 μM for DI and DI-PPA-NPs in 72 h, respectively. The superior antiproliferative activity was supported by the fact that DI-BPA-NPs could be preferentially internalized by Hela cells, owing to their specific interaction between biotin and overexpressed biotin receptors. In addition, DI-BPA-NPs effectively inhibited Hela cell proliferation by inducing G2/M phase cycle arrest and decreasing the intracellular reactive oxygen species (ROS) level by 31.50 ± 2.29% in 5 min. In summary, DI-BPA-NPs shows improved antiproliferative activity against human cervical cancer as comparing with free DI, demonstrating its application potential in cancer therapy. KEYWORDS: 15,16-dihydrotanshinone I, PLGA nanoparticles, biotin, targeted delivery, antiproliferative, cervical cancer

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activation.9 However, the anticancer effects of free DI are limited by its poor aqueous solubility and nonspecific distribution.10 Several carrier materials including liposomes,11 nanoparticles (NPs),12 microemulsions,13 and cyclodextrin inclusion14 have been developed to improve its aqueous solubility. Among carriers of these materials, NPs possess advantages in terms of solubility, biodegradability, large surface area, and permeability. PLGA is a Food and Drug Administration (FDA) approved material to prepare biocompatible and biodegradable nanoparticles.15,16 Their liphophic core offers high loading efficiency for hydrophobic therapeutic agents, which showed improved aqueous solubility, sustainable release capacity, and enhanced antitumor activity.17−19 Additionally, the hydrophilic polymer

INTRODUCTION Salvia Miltiorrhiza Bunge (Lamiaceae), known as Chinese red sage or Danshen, is one of the most commonly used medicinal plants in the traditional Chinese medicine system. It has been used as a treatment for many diseases including coronary, caridovascular, cerebrovascular, and inflammatory disease.1−4 Extracts from the dried root of Salvia Miltiorrhiza have also been developed as food supplements in China for immunitystrengthening property.5,6 15,16-Dihydrotanshinone I (DI), as one of the major bioactive compounds isolated from the root of Salvia Miltiorrhiza, has been extensively studied for anticancer activity. Tsai et al. reported that DI inhibited the proliferation of human breast cancer cell lines MCF-7 and MDA-MB-231 by inducing cell apoptosis through mitochondrial apoptosis pathways.7 Wang et al. showed that DI suppressed the growth of Hela cells in vivo by modulating the production of TNF-a.8 Ye et al. reported that DI induced cytotoxicity of human cervical cancer Hela cells by downregulation of particular human papillomavirus (HPV) E6 gene expression and caspase © 2018 American Chemical Society

Received: Revised: Accepted: Published: 9219

May 24, 2018 July 21, 2018 July 29, 2018 August 13, 2018 DOI: 10.1021/acs.jafc.8b02698 J. Agric. Food Chem. 2018, 66, 9219−9230

Article

Journal of Agricultural and Food Chemistry

methods including Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance (1H NMR) spectroscopy. 1H NMR spectra were recorded for PLGA, PPA, and BPA by a AVANCE III HD 600 NMR spectrometer (Bruker, Germany), using deuterated chloroform (CDCl3) as solvent. FTIR spectra were obtained using a Bruker VERTEX 70 IR spectrometer (Karlsruhe, Germany) over a range of 4000−500 cm−1. Preparation of DI-Loaded NPs. DI-loaded biotin-PEG-PLGA NPs (DI-BPA-NPs) were prepared using emulsion-solvent evaporation method as reported previously with some modifications.25,26 Briefly, DI (3 mg) and BPA (30 mg) were dissolved in DCM (2 mL) as the oil phase. Then, the organic solution was added into 15 mL of 1% PVA in an ice-bath and sonicated with an ultrasonic homogenizer (Misonix, NY, U.S.A.) for 5 min at 60% amplitude to obtain the O/W emulsion. The final solution was subsequently added into water (15 mL) containing 0.5% PVA and left under stirring for at least 4 h to remove DCM. The resulting solution was centrifuged at 15 000 rpm for 30 min at 4 °C, washed thrice with phosphate-buffered saline (PBS) to remove the excess of PVA and free DI, and stored at 4 °C for further use. DI-free NPs were produced in a similar method without adding DI. Characterization of DI-Loaded NPs. Particle size, size distribution, and zeta potential of NPs were measured with a Malvern Zetasizer (Malvern Instruments, Malvern, U.K.) at a temperature of 25 ± 0.5 °C and a detection angle of 90°. The samples were diluted with Milli-Q water and sonicated for several minutes before measurement. The data were obtained with the average of three independent measurements. Surface morphology of NPs was observed by a Carl Zeiss AG scanning electron microscope (SEM; Jena, Germany). A drop of NPs suspension was deposited onto aluminum stub and dried at room temperature. The samples were analyzed after being coated with gold under an argon atmosphere. To determine encapsulation efficiency and loading content of DI in DI-loaded NPs, the samples were dissolved in DCM and subjected to sonication in water bath to release encapsulated drug DI. Then, DCM was evaporated under vacuum and methanol was added to dissolve it for subsequent high-performance liquid chromatography (Agilent Technology, CA, U.S.A.). The mobile phase of DI detection was composed of pure water and methanol (20:80, v/v), and the flow rate was 0.8 mL/ min. The column temperature was maintained at 25 °C, the injection volume was 10 μL, and the UV−vis detection wavelength was set at 280 nm. The working solutions of DI with 2.5, 5, 10, 20, 40, 80, and 160 μg/ mL were prepared by serial dilution of DI stock solution. All measurements were performed in triplicate. NPs were stored at 4 °C, and particle sizes were determined by dynamic light scattering (DLS) to evaluate the stability of them.

PEG can improve the aqueous stability by decorating on the surface of PLGA-NPs. Receptor-mediated endocytosis, with the capability of targeted delivery, had attracted much attention due to the enhanced cell-uptake efficiency.20 Biotin receptors are overexpressed in tumor cells compared with normal cells. It has been demonstrated that NPs modified with biotin significantly increased cell cytotoxicity in tumor cells.21,22 DI can be efficiently embedded in PLGA-NPs with its small molecular weight and high hydrophobicity. Herein, we synthesized PPA and BPA copolymers. DI-PPANPs and DI-BPA-NPs were prepared and evaluated for their antiproliferative activity in human cervical cancer Hela cells. MTT assay and scratch wound-healing method were used to compare cell proliferation and migration after DI, DI-PPA-NPs and DI-BPA-NPs treatment. Cell cycle arrest and intracellular ROS generation were also measured to verify the antiproliferative mechanism of these DI formulations. In summary, DIBPA-NPs showed improved antiproliferative activity against human cervical cancer by comparing with free DI, demonstrating its application potential in cancer therapy.

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MATERIALS AND METHODS

Chemicals. PLGA (lactide/glycolide = 50:50; Mw = 24 000−38 000 Da) was purchased from Sigma-Aldrich (Munich, Bavaria, Germany). Biotin-PEG-NH2 and NH2-PEG-NH2 (Mw = 3 400 Da) were obtained from Peng Shuo Biotechnology (Shanghai, China). 15,16-Dihydrodanshinone I (DI, >98%) was purchased from Sen Bei Jia Biotechnology (Nanjing, Jiangsu Province, China). Anhydrous dimethyl sulfoxide (DMSO), anhydrous dichloromethane (DCM), coumarin-6 (C6), N,N-dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), triethylamine, and poly(vinyl alcohol) (PVA) were obtained from Aladdin Technology (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotic mixture (penicillin-streptomycin) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Hoechst 33342, DHE, DCFH-DA, MTT, and propidium iodide (PI) were obtained from Beyotime Biotechnology (Shanghai, China). Milli-Q water was used in all experiments. Human cervical cancer Hela cells were purchased from American Type Culture Collection (Manassas, VA, U.S.A.). Synthesis of PPA and BPA Copolymers. The PEG-PLGA (PPA) and biotin-PEG-PLGA (BPA) copolymers were prepared using standard carbodiimide method as previously described.23,24 Briefly, PLGA (500 mg, 0.02 mmol) was dissolved in DCM (5 mL) together with DCC (33.02 mg, 0.16 mmol) and NHS (18.42 mg, 0.16 mmol), and then the reactions were stirring continuously for 12 h at room temperature (RT) under nitrogen environment to obtain PLGA-NHS ester (activated PLGA). Then, the resultant solution was filtered to remove the byproduct dicylohexylurea (DCU) with a 0.45 μm polytetrafluoroethylene (PTFE) syringe filter (Millipore, Billerica, U.S.A.). The PLGA-NHS was obtained by precipitation with ice cold diethyl ether after solvents removal under vacuum. Next, the PLGA-NHS (500 mg, 0.02 mmol) was dissolved in DCM (5 mL) and added dropwise to biotin-PEG-NH2 (272 mg, 0.08 mmol) dissolved in DCM (5 mL). Triethylamine (100 μL) was added to the solution as a catalyst. Biotin-PEG-NH2 was used in excess to suppress the formation of PLGA-PEG-PLGA triblock copolymers. Then, the reaction mixture was kept on a stirrer at RT under nitrogen environment for 12 h. The reaction mixture was precipitated in ice cold diethyl ether and dried under vacuum to obtain the resultant product BPA. The collected product was dissolved in DMSO, transferred into a dialysis bag (MWCO 3.5 kDa), and dialyzed against water for 48 h to remove excess biotin-PEG-NH2. Then, the final product BPA was freeze-dried and stored at −20 °C until further use. The PPA copolymers were prepared with the same dicyclohexylcarbodiimide (DDC)/NHS chemistry method. Characterization of PPA and BPA Copolymers. The syntheses of the new polymers PPA and BPA were analyzed by spectroscopic

encapsulation efficiency (EE, %) weight of DI in nanoparticles = × 100% total amount of DI

drug loading (DL, %) =

weight of DI in nanoparticles × 100% weight of nanoparticles

In Vitro Release Studies. The release rate of DI from DI-BPA-NPs was measured by the dialysis bag method reported previously with some modifications.27 Briefly, 2 mL DI-BPA-NPs suspension that contained theoretical DI (1 mg) was added into two dialysis bags (molecular weight = 2 700), respectively. Then, the bags were immersed into centrifuge tubes containing 10 mL of release buffer (methanol/PBS = 40:60, v/v) at different pH conditions (7.4 and 5.3). Methanol was utilized to raise the solubility of DI in the release buffer. The tubes were thereafter placed in a shaker water bath and shaken at 120 rpm at 37 ± 0.5 °C. After reaching the specified time, 2 mL of release buffer was collected for analysis and replaced with an equal volume of release buffer. The content of DI was quantified by high-performance liquid chromatography (HPLC) with the method already mentioned. Cytotoxicity Assay. The cell viability was determined using the MTT assay as previously described by Zhao et al.28 Hela cells were seeded into 96-well plates at a density of 2 × 104 cells/well and cultivated at 37 °C in 5% CO2 atmosphere overnight. Then, cells were 9220

DOI: 10.1021/acs.jafc.8b02698 J. Agric. Food Chem. 2018, 66, 9219−9230

Article

Journal of Agricultural and Food Chemistry

Figure 1. Scheme for the preparation of BPA copolymer. incubated with the suspension of free DI, DI-PPA-NPs, and DI-BPANPs at equivalent DI concentrations from 2.5 to 80 μM for 24, 48, and 72 h, respectively. DI-free PLGA-NPs, PPA-NPs, and BPA-NPs at concentrations from 12.5 to 400 μg/mL were also tested. At indicated time intervals, 30 μL of MTT (5 mg/mL in PBS) was added into each well of the plates and incubated for further 4 h. Then the solution was removed and formazan crystals were dissolved by adding DMSO (150 μL/well). The mixture was shaken for 10 min. The absorbance was detected by a microplate reader (SpectroAmax TM 250, U.S.A.) at 570 nm. The untreated cells with 100% viability were used as control, and cells without MTT addition were used as blank.

cell viability (%) =

well. The concentrations of C6 in cells were measured using a fluorescence microplate reader (Spectra Max M5, Sunnyvale, CA, U.S.A.) with excitation and emission wavelengths set at 425 and 545 nm, respectively. Biotin Competing Assay. To verify the overexpression of biotin receptors on Hela cells, biotin competing assay was performed using a previously reported method.29 Briefly, Hela cells were seeded in 96-well plates at a density of 8 × 104 cells/well for 12 h. After that, they were pretreated with 100 μM biotin for 2 h before being incubated with DIPPA-NPs and DI-BPA-NPs (80 μg/mL equivalent to DI) for 8 h. Then, cells were washed three times with PBS to remove extra NPs before being lysed. The concentrations of DI in cells were measured with the excitation and emission wavelengths set at 250 and 500 nm, respectively. In addition, a standard curve of DI was generated with a concentration of 80−0.625 μg/mL in the same 96-well plates. Cell Cycle Analysis. The cell cycle distribution was measured by flow cytometric analysis reported previously.29 Briefly, Hela cells were harvested after treatment with 5, 10, and 20 μM DI and DI-BPA-NPs (5, 10, and 20 μM equivalent to DI) for 24 h, washed with PBS three times, and fixed with 70% cold ethanol at −20 °C overnight. The fixed cells were then stained with 500 μL of PI for 30 min in the dark. The stained cells were measured using a flow cytometer with Cell Quest software (Beckman Coulte, FL, U.S.A.). For all experiments, 10 000 events per sample were recorded. The Multi Cycle software (Phoenix Flow Systems, CA, U.S.A.) was used to analyze the cell cycle distribution. Cell Migration Assay. Cell migration was evaluated by scratch wound-healing method according to the literature methods.30 Briefly, Hela cells were seeded at a density of 20 × 104 cells/mL and cultured in 6-well plates overnight. A scratch wound was created by a sterile pipet tip. Then the culture medium was aspirated and cells were washed with PBS three times before being incubated with 3% FBS fresh medium for 4 h. Nuclei were stained with 2 μL of Hoechst 33342 for 30 min. After

Abs(sample) − Abs(background) × 100% Abs(control) − Abs(background)

Cellular Uptake of C6-Loaded NPs. To investigate the cellular uptake of the NPs, coumarin-6 (C6) was used as a fluorescent probe and C6-loaded NPs were prepared by the same method as described for DI-loaded NPs. The qualitative uptake of C6-loaded NPs in Hela cells was determined by measurement of fluorescence intensity in cells. Briefly, Hela cells were seeded in a six-well plate at a density of 2 × 104 cells/mL for 24 h. Then the media was substituted with fresh media containing C6-PPA-NPs and C6-BPA-NPs carrying equivalent concentrations of C6, respectively. After incubation for 4 h at 37 °C, cells were washed three times with PBS to remove unabsorbed NPs and were examined under a fluorescence microscope. For quantitative analysis, Hela cells were seeded in 96-well plates at a density of 8 × 104 cells/well for 12 h, and then they were incubated with C6-PPA-NPs and C6-BPA-NPs (80 μg/mL equivalent to C6) for 8, 12 and 24 h, respectively. After reaching the specified time, the medium was removed and cells were washed three times with PBS to remove C6-loaded NPs outside cells. After that, cells were lysed by adding 0.1 M NaOH solution containing 1% Triton X-100 (200 μL) into each 9221

DOI: 10.1021/acs.jafc.8b02698 J. Agric. Food Chem. 2018, 66, 9219−9230

Article

Journal of Agricultural and Food Chemistry

Figure 2. Chemical structure of PPA and BPA. (A) FTIR spectra of NH2-PEG-NH2, biotin-PEG-NH2, PLGA, PPA, and BPA. (B) 1H NMR spectra of PLGA, PPA, and BPA. that, cells were photographed immediately. Then, cells were incubated

cell mobility (%) =

with 8 μM DI and DI-BPA-NPs (2, 4, and 8 μM equivalent to DI) for 24

number of migrated cells in treated group number of migrated cells in control group × 100%

Measurement of the ROS. The intracellular ROS generation was carried out according to the method described previously with some modifications.31 Briefly, Hela cells were seeded in 96-well plates at a

h before being dyed by Hoechst 33342 and photographed under a fluorescence microscope. 9222

DOI: 10.1021/acs.jafc.8b02698 J. Agric. Food Chem. 2018, 66, 9219−9230

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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Characterizations of PLGA-NPs, DI-PLGA-NPs, DI-PPA-NPs, and DI-BPA-NPsa formulation

size (nm)

PDI

zeta (mV)

EE (%)

DL (%)

PLGA-NPs DI-PLGA-NPs DI-PPA-NPs DI-BPA-NPs

153 ± 1.65 169 ± 2.73 180 ± 3.12** 179 ± 4.01**

0.131 ± 0.0121 0.151 ± 0.0162 0.124 ± 0.0183 0.143 ± 0.0139

−12.9 ± 1.06 −14.6 ± 1.21 −16.7 ± 1.55 −26.7 ± 1.79

0 70.1 ± 6.16 68.1 ± 8.18 67.5 ± 9.14

0 6.58 ± 0.434 6.35 ± 0.742 6.24 ± 0.833

**P < 0.01, compared with DI-PLGA-NPs.

a

Figure 3. Properties of the NPs. (A) SEM image of DI-BPA-NPs; (B) zeta potentials of DI-BPA-NPs; (C) particle sizes and PDI of DI-BPA-NPs; (D) stability of DI-BPA-NPs in a week. Values expressed are means ± SD of triplicates. density of 20 × 104 cells/well for 12 h. Then the media was removed and replaced with fresh media containing 10 μM DHE and DCFH-DA. After being incubated for 30 min, cells were treated with DI, DI-PPANPs, and DI-BPA-NPs at equal DI concentrations. The generation of ROS was then detected within 2 h by the fluorescence intensity. The excitation and emission wavelengths of DHE were set at 300 and 610 nm, respectively. The excitation and emission wavelengths of DCFHDA were set at 488 and 525 nm, respectively. The ratio of fluorescence intensity in the treated and control groups were calculated, and the changes of intracellular ROS were analyzed. Images were taken under a fluorescence microscope. Statistical Analysis. All the experiments were performed at least three times. The experimental data were expressed as mean ± SD. Statistical analysis was performed using SPSS statistical package (SPSS 17.0 for Windows; SPSS, Inc., Chicago, IL, U.S.A.). The difference between two groups was analyzed by Student’s t test. The difference between three or more groups was analyzed by one-way analysis of variance. The difference between groups was statistically significant (*P < 0.01 or **P < 0.05). Bars with different characters are statistically different at P < 0.05 level.

Figure 4. In vitro release profile of DI from DI-BPA-NPs in PBS solution at pH 7.4 or at pH 5.3, respectively. Values expressed are means ± SD of triplicates.

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conjugated to PLGA via standard carbodiimide method using DCC/NHS as an activating agent. The preparation of DI-BPANPs is shown in Figure 1. As shown in Figure 2A, the conjugations between biotin and NH2-PEG-NH2 were confirmed by the presence of new peaks that appeared at 1688.2 and

RESULTS AND DISCUSSION Rational Design and Characterization of PPA and BPA Copolymer. NH2-PEG-NH2 and biotin-PEG-NH2 were 9223

DOI: 10.1021/acs.jafc.8b02698 J. Agric. Food Chem. 2018, 66, 9219−9230

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Journal of Agricultural and Food Chemistry

Figure 5. Cell viability of Hela cells after treatment with DI-free NPs for 72 h (A) and three DI formulations for 24 h (B), 48 h (C), and 72 h (D) at various concentrations. Values expressed are means ± SD of triplicates. Bars between DI-loaded NPs groups and free DI groups are significantly different at P < 0.05 (*) or P < 0.01 (**) levels.

the −CH2− group in the glycolide unit, and peaks at 1.57 and 5.20 ppm correspond to the methyl group and −CH− group in the D,L-lactide unit. The major difference of these copolymers was the peak at 3.65 ppm corresponding to the −OCH3 group and the methylene group in the PEG unit. This observation confirmed the connection of PEG and PLGA. Nanoparticles Characterization. In this study, the NPs were prepared using an emulsion solvent evaporation method. Particle size and size distribution are important factors for the development of suitable nanomedicines.32 As shown in Table 1, DI-loaded PLGA-NPs showed a particle size of 169 ± 2.73 nm that was bigger than that of DI-free PLGA-NPs of 153 ± 1.65 nm. This could be attributed to the load of hydrophobic DI into the NPs, which changed the structure and increased the particle size of PLGA-NPs.33 On the other hand, with the PEG decorated on the surface of the NPs, the particle size of DI-PPANPs and DI-BPA-NPs increased up to 180 ± 3.12 nm and 179 ± 4.01 nm, respectively, which were larger than that of DI-PLGANPs. However, no significant difference between the size of DIPPA-NPs and DI-BPA-NPs was detected, suggesting that the biotin on the surface of the PEG did not influence the thickness of the PLGA shell. Taken together, all formulations had relatively desirable particle sizes (