Metformin and histone deacetylase inhibitor based anti-inflammatory nanoplatform for epithelial-mesenchymal transition suppression and metastatic tumor treatment | Journal of Nanobiotechnology

[ad_1]

Materials

Propofol, dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), butylated hydroxytoluene (BHT), anhydrous dichloromethane, dextran sulfate and phosphotungstic acid were purchased from Aladdin Co., Ltd (Shanghai, China). Docosahexaenoic acid (DHA), coumarin-6, 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl indotricarbocyanine iodide (DiR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pyrene was provided by J&K scientific Co., Ltd (Shanghai, China). Triptolide was purchased from Biopurify Phytochemicals Ltd (Chengdu, China). HA (MW = 30,000) was provided by Bloomage Biotechnology Co., Ltd (Jinan, China). D-luciferin potassium was bought from PerkinElmer Inc. (Waltham, MA, USA). Interleukin-13 (IL-13) was purchased from Sinobio Co., Ltd (Shanghai, China). Matrigel was acquired from BD Bioscience (San Jose, CA, USA). Crystal violet staining solution was obtained from Beyotime Biotechnology Co., Ltd (Shanghai, China). Phycoerythrin (PE) labeled anti-CD24 antibody, PE labeled anti-CD206 antibody and fluorescein isothiocyanate (FITC) labeled anti-CD44 antibody were provided by eBioscience. Anti-Histone H3 (acetyl K9) antibody, anti-E-cadherin antibody, anti-MMP-9 antibody and anti-Collagen I antibody were bought from Abcam (Cambridge, MA, USA). Anti-phospho-AMPK (p-AMPK, Thr172) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). In situ cell death detection kit was obtained from Roche (Basel, Switzerland). All the other chemical reagents and solvents were acquired from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Dulbecco’s modified eagle medium (DMEM), RPMI 1640 medium, certified fetal bovine serum (FBS), penicillin–streptomycin stock solutions and trypsin–EDTA (0.25%) were obtained from Invitrogen (Carlsbad, CA, USA). DMEM/F12 medium, B-27 supplement (50 ×), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were provided by Gibco (USA).

Cell lines and animals

Murine RAW264.7 macrophages cells, 4T1 cells and 4T1-Luc cells were provided by Chinese Academy of Sciences Cell Bank (Shanghai, China). RAW264.7 cells were cultured in DMEM medium, 4T1 cells and 4T1-Luc cells were cultured in RPMI 1640 medium, supplemented with 10% FBS (v/v), 100 mg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified atmosphere with 5% CO2.

Female Balb/c mice (20 ± 2 g) and female Spraguee-Dawley (S-D) rats (200 ± 10 g) were purchased from BK Lab Animal Ltd. (Shanghai, China) and kept under standard housing condition (25 ± 1 °C with access to food and water ad libitum).

Synthesis and characterization of pro-DHA

DHA (0.152 mmol) was firstly dissolved in anhydrous dichloromethane (5 mL). DCC (0.228 mmol), DMAP (0.138 mmol) and BHT (0.8 μM, an antioxidant) were added to the solution of DHA in dichloromethane, and the reaction mixture was stirred at room temperature for 2 h under nitrogen. Then a solution of propofol in anhydrous dichloromethane (0.138 mmol) was added dropwise, and the reaction mixture was stirred at reflux for 12 h under nitrogen. After the completion of reaction, the solvent was evaporated in vacuo, and the crude product was purified by column chromatography to obtain colorless oil (pro-DHA) with a yield of 53.8%. The structure of pro-DHA was verified by 1H-NMR (600 MHz, BRUKER) in CDCl3 and electron spray ionization-mass spectrometry (ESI–MS).

Preparation and characterization of HAOPTs

OPTs were first prepared by self-assembly. Briefly, pro-DHA (2 mg) and triptolide (420 μg) were dissolved in a solution of OA-Met in ethanol (500 μL at 10 mg/mL), and the final solution was added dropwise into distilled water (1 mL) under stirring (500 rpm). The suspension was stirred for another 3 h at room temperature and ethanol was absolutely removed in vacuo at 25 °C to obtain OPTs (7.42 mg/mL). Afterward, HAOPTs were prepared by electrostatic adsorption of HA on the surface of OPTs. Briefly, aqueous OPTs suspension (216 μL) was added into HA solution (2 mL, 0.8 mg/mL) dropwise under stirring (500 rpm). The suspension was stirred for another 2 h at room temperature and HAOPTs was finally obtained.

Coumarin-6 and DiR labeled OPTs, DexOPTs or HAOPTs were prepared by the same method, whereas coumrin-6 or DiR was substituted for triptolide to dissolve in the solution of pro-DHA and OA-Met. In addition, the preparation of HAOPs (HAOPTs without triptolide) and HAOTs (HAOPTs without pro-DHA) was similar to the protocol of HAOPTs, where pro-DHA or triptolide alone was dissolved in OA-Met ethanol solution for subsequent micelle preparation.

Z-average diameter, polymer dispersity indexes (PDI) and zeta potential of micelles (OPTs, DexOPTs and HAOPTs) were detected by a dynamic light scattering detector (DLS) (Zetasizer, Nano-ZS, Malvern, UK). The stability of these micelles was determined in phosphate buffer saline (PBS) at 4 °C for 12 days and the z-average diameter was recorded at different time points.

Encapsulation efficiency (EE) and loading capacity (LC) were investigated through the high performance liquid chromatography (HPLC) method. OPTs, DexOPTs and HAOPTs were dissolved in methanol and then subjected to HPLC system. Triptolide was detected at the detector wavelength of 220 nm, and the mobile phase was a mixture of methanol and distilled water (the volume ratio of methanol to water was 45:55). Moreover, pro-DHA was identified at the detector wavelength of 214 nm, and the mobile phase was the gradient elution of distilled water and methanol, which was from 80% (methanol) to 100% (methanol) in 40 min. The EE and LC were calculated by Eqs. 1 and 2 (n = 3):

$${\text{EE}}\left( \% \right) = \frac{{{\text{amount}}\,{\text{of}}\,{\text{pro}} – {\text{DHA}}/{\text{triptolide}}\,{\text{in}}\,{\text{the}}\,{\text{micelles}}}}{{{\text{total}}\,{\text{amount of}}\,{\text{pro}} – {\text{DHA}}/{\text{triptolide}}\,{\text{added}}}} \times 100\%$$

(1)

$${\text{LC}}\left( \% \right) = \frac{{{\text{amount}}\,{\text{of}}\,{\text{pro}} – {\text{DHA}}/{\text{triptolide}}\,{\text{in}}\,{\text{the}}\,{\text{micelles}}}}{{{\text{micelles}}\,{\text{weight}}}} \times 100\%$$

(2)

The critical micelle concentration (CMC) of HAOPTs was investigated by the fluorescence probe pyrene. HAOPTs with different concentrations (1 × 10–4 to 2 × 10–1 mg/mL) were added to the tubes containing pyrene (12.5 μg), then the tubes were placed in a shaker at 37 °C for 24 h (120 rpm). After scanning pyrene by a fluorescence spectrophotometer (excitation wavelength: 335 nm, emission wavelength: 373 and 384 nm), the CMC of HAOPTs was calculated by the cross point in the plots of the fluorescence intensity ratio of 384 nm and 373 nm to the logarithm concentration of HAOPTs.

The morphology of OPTs and HAOPTs was observed by a field emission transmission electron microscope (TEM, TEM-1400 Plus Electron Microscope, Leica). Aqueous OPTs and HAOPTs suspensions (1 mg/mL) were dropped on the carbon-coated grid and water was dried under a drying light. Phosphotungstic acid solution (2%, w/v) was dropped on the dried micelles for negative staining. After 5 min, the solution was removed and the grid was subjected to a TEM for photographing.

Cellular uptake of HAOPTs

Mouse breast tumor 4T1 cells were seeded into 96-well plates at a density of 3,000 cells per well firstly (n = 3). After incubation for 24 h, the cell culture media was replaced by free coumarin-6, coumarin-6 labeled OPTs-Cou, DexOPTs-Cou and HAOPTs-Cou (100 ng/mL for coumarin-6) and cells were incubated for 2 h in the dark. The cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde (PFA) for 10 min and stained with Hoechst 33,258 at room temperature for 10 min in dark places. Finally, the cells were washed with PBS buffer for another three times, subjected to an inverted fluorescence microscope (Leica DMI 4000B, Germany) for qualitative imaging, and cellular uptake achieved quantitative analysis through a Kinetic Scan HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA, USA).

Pharmacokinetic study of HAOPTs

The pharmacokinetic study of HAOPTs was investigated through the following protocol. Twelve S-D rats were divided into four groups randomly (n = 3) and intravenously injected with free DiR, DiR labeled OPTs-DiR, DexOPTs-DiR and HAOPTs-DiR (270 μg/kg for DiR), respectively. Blood samples (300 μL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h and then centrifuged at 4000 rpm for 10 min instantly. The supernatant plasma was separated from the centrifugal blood and plasma samples were stored at −20 °C.

In order to determine DiR concentration in plasma, methanol (900 μL) was added to plasma samples (100 μL) to precipitate proteins. The mixtures were vortexed for 5 min and centrifuged at 10,000 rpm for 10 min. The supernatant was separated and then subjected to microplate reader (Thermo Multiskan MK3, USA) for DiR fluorescence analysis (excitation wavelength: 748 nm, emission wavelength: 780 nm). Finally, the pharmacokinetic parameters could be calculated by Drug and Statistics (DAS) software (Version 2.0, Mathematical Pharmacology Professional Committee of China).

Biodistribution of HAOPTs in orthotopic breast tumor mice

The distribution study of HAOPTs was performed to verify the orthotopic tumor targeting effect. 4T1 cells were inoculated into one mammary fat pad of Balb/c mice. Once the tumor grew to 100 mm3–200 mm3, the mice were divided into three groups randomly (n = 3) and injected i.v. with OPTs-DiR, DexOPTs-DiR and HAOPTs-DiR (1 mg/kg for DiR). The biodistribution of DiR labeled micelles was monitored by an in vivo IVIS spectrum imaging system (Cailper PerkinElemer, USA) at 2, 6, 12 and 24 h. After the final detection, mice were euthanized and perfused with saline and 4% PFA, followed by the harvest of major organs (heart, liver, spleen, lung and kidney) and orthotopic tumors for ex vivo imaging and semi-quantitative analysis of DiR fluorescence.

MTT assay and combination index (CI) calculation

The tumor anti-proliferation activity of HAOPTs was assessed by MTT assay. Briefly, 4T1 cells were seeded into 96-well plates at the cell density of 1,000 cells per well (n = 3) and incubated for 24 h. Cells were then exposed to metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs with various concentration gradients. After incubation for 24 h, MTT solution (20 μL, 5 mg/mL) was added directly, and cells were incubated for another 4 h. The supernatant in well plates was substituted by DMSO (150 μL) to dissolve formazan crystals and the well plates were vibrated for 10 min. At last, well plates were subjected to microplate reader (Thermo Multiskan MK3, USA) for cell viability analysis at a wavelength of 570 nm and IC50 calculation.

The CI of OA-Met, pro-DHA or triptolide in micelles could be calculated by IC50 values based on Chou-Talalay theory and Eq. 3 [38, 39]:

$${\text{CI}} = \frac{{{\text{IC}}_{{50}} {\mkern 1mu} {\text{of}}{\mkern 1mu} {\text{drug}}{\mkern 1mu} \,{\text{A}}{\mkern 1mu} \,{\text{in}}\,{\mkern 1mu} {\text{combination}}\,{\mkern 1mu} {\text{therapy}}}}{{{\text{IC}}_{{50}} {\mkern 1mu} {\text{of}}\,{\mkern 1mu} {\text{drug}}{\mkern 1mu} \,{\text{A}}\,{\mkern 1mu} {\text{alone}}}} + \frac{{{\text{IC}}_{{50}} {\mkern 1mu} {\text{of}}{\mkern 1mu} {\text{drug}}\,{\text{B}}\,{\mkern 1mu} {\text{in}}\,{\mkern 1mu} {\text{combination}}{\mkern 1mu} \,{\text{therapy}}}}{{{\text{IC}}_{{50}} \,{\text{of}}{\mkern 1mu} {\text{drug}}{\mkern 1mu} \,{\text{B}}{\mkern 1mu} \,{\text{alone}}}} + \ldots$$

(3)

Mammosphere formation assay and mammosphere formation efficacy (MSFE) calculation

The mammosphere formation assay was performed as follows. Briefly, 4T1 cells were seeded into ultra-low attachment 24-well plates (2 × 104 cells/cm2) with DMEM/F12 culture medium containing B-27 Supplement (1 ×), EGF (20 ng/mL), bFGF (20 ng/mL), 100 mg/mL streptomycin and 100 U/mL penicillin (n = 3). Meanwhile, cells were treated with PBS buffer, metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (10 ng/mL for triptolide). After 5 days of incubation for sphere formation, spheres were subjected to an inverted microscope (Leica DMI 4000B, Germany) for imaging and counting, and MSFE was calculated by following Eq. 4:

$${\text{MSFE}}\left( \% \right) = \frac{{{\text{number of spheres}}\,({\text{diameter greater than 50 }} \mu {\text{m}})}}{{\text{number of cells seeded }}} \times 100\%$$

(4)

Flow cytometry assay for analysis of cancer stem cells in vitro

The phenotype of breast cancer stem cells (CD44+/CD24−/low) was identified by flow cytometry. After the mammosphere formation assay (n = 3), the cell suspensions were centrifuged at 800 rpm for 3 min to collect the spheres. Spheres were then treated with trypsin–EDTA (0.05%) for 3 min and centrifuged at 800 rpm for 3 min instantly. Additionally, single cells were collected, washed with PBS buffer (1 mL) and centrifuged at 1700 rpm for 5 min. After washing with PBS buffer for another two times, cells were stained with PE labeled anti-CD24 antibodies and FITC labeled anti-CD44 antibodies simultaneously away from light at 4 °C for 30 min. Finally, the stained cells were washed for three times and resuspended in PBS buffer (200 μL) for flow cytometric analysis by a BD FACScan (BD FACSAria II).

Macrophages polarization assay in vitro

To investigate the ability of HAOPTs in the inhibition of M2 macrophage polarization, RAW264.7 cells were seeded into 6-well plates at the cell density of 1 × 105 cells per well (n = 3). Macrophages were then stimulated with IL-13 (10 ng/ml) for 24 h followed by treating with PBS buffer, metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (10 ng/mL for triptolide). After incubation for 24 h, cells were washed with PBS buffer for three times and collected by centrifuging at 1000 rpm for 4 min. Macrophages were then stained with PE labeled anti-CD206 antibodies in the dark at 4 °C for 30 min. At last, the stained cells were washed for three times and CD206-positive M2 macrophages were detected by flow cytometry (BD FACSAria II).

Tumor cell invasion assay

The tumor cell invasion assay was performed through Transwell systems. Transwell membranes (8 μm pore size, Corning Costar Co., Cambridge, MA, USA) were coated with Matrigel and incubated at 37 °C for 4 h. The reconstituted Matrigel coatings were washed with RPMI 1640 twice and soaked for 30 min in RPMI 1640. 4T1 cells were suspended in RPMI 1640 containing 1% FBS (1 × 106 cells/mL). The cell suspensions (100 μL) were seeded into the upper wells of coated Transwells and treated with PBS buffer, metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (10 ng/mL for triptolide, final volume was 200 μL). Lower wells of the transwells were filled with RPMI 1640 containing 10% FBS, and PBS buffer, metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (10 ng/mL for triptolide) were added to the lower wells (final volume was 500 μL). After incubation for 24 h, membranes coated with Matrigel were fixed with methanol at − 20 °C for 15 min, swabbed with a cotton swab and washed with PBS buffer for three times. Once the membranes were died, cells on the membranes were stained with crystal violet solution (500 μL) at 37 °C for 30 min. The transwells were then washed with PBS buffer and imaged under an inverted fluorescence microscope (Leica DMI 4000B, Germany). Afterward, the transwells were soaked in 33% acetic acid solution (500 μL) for 10 min, and the number of invasive cells was determined through the absorbance of crystal violet recorded by a microplate reader (Thermo Multiskan MK3, USA) at a wavelength of 570 nm.

Immunohistochemical staining for orthotopic tumor sections

4T1 cells were inoculated into one mammary fat pad of Balb/c mice. On the sixth day after tumor inoculation, the mice were divided into nine groups randomly (n = 4) and infused with saline, metformin, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (0.7 mg/kg for triptolide) one time every two days via tail veins. Two days after the last injection (day 16), mice were euthanized and perfused with saline and 4% PFA, and tumors were harvested, fixed and embedded in paraffin. Moreover, the sections of tumors were applied for anti-mouse p-AMPK, Histone H3 (acetyl K9), E-cadherin, MMP-9, Collagen I antibodies and HRP double staining. The stained sections were finally subjected to an inverted fluorescence microscope (Leica DMI 4000B, Germany) for photographing and quantitative analysis was completed by using ImageJ 1.46 version program.

Therapeutic efficacies of HAOPTs in orthotopic breast tumor mice

We adopted 4T1-Luc cell-derived orthotopic breast tumor mice to demonstrate the inhibitory effects of orthotopic breast tumor growth and spontaneous metastasis by HAOPTs. Briefly, 4T1-Luc cells were inoculated into one mammary fat pad of Balb/c mice. Once the volume of orthotopic tumors reached about 100 mm3, the mice were divided into eight groups randomly (n = 10) and intravenously injected with saline, OA-Met, pro-DHA, triptolide, HAOPs, HAOTs, DexOPTs and HAOPTs (0.7 mg/kg for triptolide) once every three days. The orthotopic tumor volumes were measured by a Vernier caliper during the therapeutic process. After all injections (day 27), five random mice from each group were euthanized and perfused with saline, and major organs (heart, liver, spleen, lung and kidney) and tumor tissues were collected. Additionally, tumors were weighed, fixed and embedded in paraffin, and tumor sections were stained with hematoxylin & eosin (H&E) or in situ cell death detection kit (TUNEL assay) followed by analysis of necrosis or apoptosis under an inverted fluorescence microscope (Leica DMI 4000B, Germany).

Furthermore, the metastasis suppression efficacy was evaluated by following methods. Harvested lung tissues were instantly soaked in D-luciferin solution (0.5 mg/mL) for 5 min, and then detected by an in vivo IVIS spectrum imaging system to record bioluminescence images and bioluminescent quantitative data. Besides, the rest five mice in each group were performed for the survival study.

Toxicity evaluation of HAOPTs

In order to verify the safety of HAOPTs, the weight of mice was examined every three days during the above therapy. After all treatments (day 27), harvested major organs (heart, liver, spleen, lung and kidney) were served for H&E staining and imaging. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels in the serum were detected.

Statistical analysis

All data were displayed as the mean ± standard deviation (SD). Student’s t-test was served for a difference between two groups, and the comparisons among multiple groups were analyzed by one-way ANOVA in Graphpad Prism 6.02. Statistical significance was presented with P value lower than 0.05.

[ad_2]

Source link

Leave a Reply

Your email address will not be published.