Carboxymethyl Dextran-Stabilized Polyethylenimine-Poly(epsilon

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Carboxymethyl Dextran-Stabilized PolyethyleneiminePoly(epsilon-caprolactone) Nanoparticles-Mediated Modulation of MicroRNA-34a Expression via SmallMolecule Modulator for Hepatocellular Carcinoma Therapy Xiongwei Deng, Zhaoxia Yin, Zhixiang Zhou, Yihui Wang, Fang Zhang, Qin Hu, Yishu Yang, Jianqing Lu, Yan Wu, Wang Sheng, and Yi Zeng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03122 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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ACS Applied Materials & Interfaces

Carboxymethyl Dextran-Stabilized Polyethyleneimine-Poly(epsilon-caprolactone) Nanoparticles-Mediated Modulation of MicroRNA-34a Expression via Small-Molecule Modulator for Hepatocellular Carcinoma Therapy Xiongwei Deng, ⊥, †,‡ Zhaoxia Yin, ⊥,† Zhixiang Zhou, † Yihui Wang, † Fang Zhang, † Qin Hu,a Yishu Yang, † Jianqing Lu, ‡ Yan Wu,*,‡ Wang Sheng*,† and Yi Zeng† †

College of Life Science and Bioengineering, Beijing University of Technology, No. 100 Pingleyuan, Chaoyang District, Beijing 100124, P.R. China.

‡

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for

Excellence in Nanoscience, National Center for Nanoscience and Technology, No. 11 Beiyitiao, Zhongguancun, Beijing 100190, China

KEYWORDS: Carboxymethyl dextran; Polyethyleneimine-Poly(epsilon-caprolactone); Nanoparticles; MicroRNA modulation; Small-molecule modulator; Hepatocellular carcinoma

ABSTRACT MicroRNA-34a (miR-34a) modulation therapy has shown great promise to treat hepatocellular carcinoma (HCC). 2'-hydroxy-2,4,4',5,6'-pentamethoxychalcone termed Rubone, has been shown to specifically up-regulate miR-34a expression in HCC cells and considered as novel anti-cancer agent. However, the extremely low aqueous solubility of Rubone hampers its use in cancer treatment. In the present study, surface-stabilized nanoparticles-based delivery strategy was engaged to overcome this impediment. In our preparation, Rubone was encapsulated in the micelles

composed

of

polyethyleneimine-poly(epsilon-caprolactone)

(PEI-PCL)

through

hydrophobic interactions, which were subsequently stabilized with anionic carboxymethyl dextran CMD via electronic interaction. We found that Rubone-encapsulating nanoparticles are dispersed well in aqueous solution. The results further demonstrated that Rubone could be efficiently delivered in HCC cells by nanoparticles and up-regulate miR-34a expression, which in turn led to

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inhibition of proliferation, migration and increased apoptosis of HCC cells. In vivo experiments showed that Rubone can be preferentially delivered into tumor tissues by CMD-stabilized PEI-PCL nanoparticles after intravenous administration and significantly inhibited tumor growth. Furthermore, low cytotoxicity of the nanoparticles was observed in vitro and in vivo analyses, indicating a good compatibility of generated nanoparticles. The obtained results suggest that CMD-stabilized

PEI-PCL

nanoparticles

may

serve

as

novel

approach

for

small

molecule-modulator-mediated miR-34a restoration for HCC therapy.

1. INTRODUCTION Human hepatocellular carcinoma (HCC) is one of the most common malignancies that threaten human health.1 At early stages, liver transplantation, surgical resection and thermal ablation are potentially curative interventions for HCC. Unfortunately, most of the patients are diagnosed with advanced HCC, where curable approaches are often not feasible. Traditional chemotherapy was used to treat HCC for over 30 years, although survival benefit are limited.2 Although sorafenib has been approved to treat advanced HCC, the overall survival time after treatment with sorafenib is still limited (from a median of 8 months to 11 months).3 Hence there is an urgent need to identify HCC-specific drug targets and to develop novel therapeutics. MicroRNAs (miRNAs) are highly conserved and small non-coding RNA that regulate gene expression at the post-transcriptional level.4 Aberrant expression of miRNAs has been discovered in most cancers, acting as either tumor suppressors or oncogenes. In general, down-regulated miRNAs were recognized as tumor suppressors while up-regulated miRNAs were recognized as oncogenes. MicroRNA-34a (miR-34a) is a well-defined tumor suppressor in many tumor types and has been recognized as a key regulator of tumor progression.5,6 Several cancer-related signaling pathways can be modulated by miR-34a, including the p53 and wnt/β-catenin pathways and the restoration of miR-34a expression could inhibit the growth and progression of many tumors.7 Restoration of miR-34a level through nucleic acid-based tools including miR-34a mimics and plasmid expression vectors are the most commonly used methods currently.8,9 Different nanoparticle-based non-viral delivery platforms have been developed for miRNA delivery both in vitro and in vivo.10-12 For instance, MRX34 (Mirna Therapeutics, Austin, TX), a nanosized


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miR-34a mimic-loaded liposome, has entered phase 1 clinical trial to treat primary liver cancer.13 Although non-viral-based vectors for nucleic acid-based miRNA modulators have been truly exciting, several crucial problems associated still limit its application, such as low bioavailability in vivo, enzymatic degradation, activation of immune responses and high cost of treatment.14-17 For this reason, there are great needs to explore new alternative methods to regulate aberrant miRNA expression for cancer therapy. In recent years, small molecule modulators of miRNA expression have been discovered. Small molecules are potentially better candidates than nucleic acid-based drugs since they can be more stable to overcome degradation by serum nucleases and they can be are more easily delivered both in vitro and in vivo.18 In general, small molecules can modulate the expression of miRNAs by either activating or repressing their transcription. They could provide versatile tools for the elucidation of mechanisms of miRNA function and regulation, and serve as novel therapeutics.19 Recently, a natural compound 2'-hydroxy-2,4,4',5,6'-pentamethoxychalcone termed Rubone was discovered by a miR-34a luciferase reporter system using HCC cells, which was able to enhance mature miR-34a expression by effectively activating the synthesis of pri-miR-34a.20 In vitro and in vivo studies have shown effective anti-HCC activities of Rubone through up-regulation of miR-34a expression, thereby decreasing the expression and function of target oncogenes.21 However, its poor aqueous solubility limited its application through systemic administration. During the past few decades, nanoparticle drug formulations have been intensively investigated to deliver insoluble drugs to tumor sites and cells efficiently.22 Different nanoparticle delivery systems, including inorganic nanoparticles, polymeric micelles, liposomes, polymer-drug conjugates and metal nanoclusters, have proved to be promising drug-delivery systems owing to their good biocompatibility, controlled-releasing property, lower immunogenicity and ease of production and functionality.23-26 Moreover, nanoparticles have been extensively investigated for selective passive targeting ability as the specific “leaky” structure of tumor vasculature (enhanced permeability and retention [EPR] effects),

which

was

first

proposed

by

Hiroshi

Maeda

demonstrating

that

the

high-molecular-weight therapeutic agent naming SMANCs could enhance accumulation in solid tumor sites.27,28 To increase in vivo circulation time and enhance accumulating of nanoparticles at the tumor site by passive EPR effect, the nanoparticle surface is usually stabilized by hydrophilic materials



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to reduce plasma opsonization and uptake of monocyte phagocytic system.29

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The common

method include successfully creating “stealth” brushes by coating the surface of nanoparticles using polyethylene glycol (PEG), polyvinyl alcohol (PVA), zwitterionic materials and biocompatible polysaccharides.30-32 Carboxymethyl dextran (CMD) is an anionic dextran derivative. Due to its good biocompatibility, CMD has been widely used for many biomedical applications, including drug and gene delivery.33-35 CMD coating on nanoparticles has shown excellent resistance to nonspecific protein adsorption due to their strong hydration via hydrogen bonding.36 In addition, CMD coating has been used in many interfacial biosensor detectors.37 Polyethyleneimine-poly(epsilon-caprolactone) (PEI-PCL) was an amphiphilic diblock copolymer that has been widely used as drug/gene delivery38,39. The hydrophobic block PCL was an FDA-approved material and could serve as the inner core encapsulating hydrophobic drugs efficiently through hydrophobic interactions. The abundant amine groups in PEI could promote endosomal rupture via “proton sponge effects”.40 In general, low molecular weight of PEI shows better biocompatibility than high molecular weight of PEI, while exhibiting equivalent buffering capacity.41 In addition, this copolymer could provide ease of surface functionalities due to its active amine groups. In

this

study,

carboxymethyl

dextran-stabilized

polyethyleneimine-poly(epsilon

-caprolactone) nanoparticles (RC-NPs) were prepared to encapsulate and deliver a small molecule-modulator of miR-34a Rubone for HCC therapy (Scheme 1). Rubone was firstly encapsulated in the inner core of PEI-PCL nanoparticles and then coated with an anionic CMD layer via electronic interactions. The nanoparticle size, surface zeta potential, morphology, Rubone encapsulation and loading efficiency, in vitro release profiles of Rubone, colloidal stability and buffering capability of the nanoparticles were characterized. The subcellular location of Cy-3 fluorescence labeled RC-NPs in HepG-2 cells was analyzed by confocal laser scanning microscopy. We therefore investigated the miR-34a modulation effects, anti-HCC effects and mechanisms of Rubone-loaded RC-NPs through in vitro/in vivo models. Furthermore, in vivo biodistribution and systemic biocompatibility of RC-NPs after intravenous administration were also studied.

2. EXPERIMENTAL SECTION


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2.1. Materials. 2'-hydroxy-2,4,4',5,6'-pentamethoxychalcone was purchased from International Laboratory USA. Dextran (Mw=27 KDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Amphiphilic PEI-PCL copolymer (Molecular weight (Mw) of PEI=800 Da, Mw of PCL=20 KDa) was synthesized and provided by Jinan DaiGang Biomaterials Company (Shandong, China). This copolymer was used without further puriﬁcation. Cell counting kit-8 was obtained from Dojindo Co. LTD. (Tokyo, Japan). Cyanine 3 monosuccinimidyl ester (Cy-3-NHS) was purchased from AAT Bioquest Inc (Sunnyvale, CA). Trizol reagent, 4’,6-diamidino-2-phenylindole (DAPI) and LysoTracker green was purchased from Invitrogen Co. LTD (USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco BRL (Grand Island, NY, USA). Other chemical reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The water used was of ultrapure grade and was supplied by a Milli-Q purification system (Millipore Co., Billerica, MA, USA). 2.2. Synthesis of carboxymethyl dextran (CMD) Carboxymethyl dextran (CMD) was synthesized from dextran according to previous studies (Scheme S1).42 In brief, 1.0 g dextran was dissolved in sodium hydroxide solution (30 mL, 2.0 M). 0.41 g bromoacetic acid was added to the above solution. The reaction was performed at room temperature for 24 h. After 24 h, the resulting mixtures were transferred into a dialysis bag and dialyzed against deionized (DI) water for 24 h, then against 0.1 M hydrochloric acid for 24 h and against DI water for 48 h again to remove residual reagents. After the dialysis, the dialyzed solution was lyophilized to yield dried CMD. The degree of substitution of carboxymethylation (the number of carboxyl group to the anhydroglucose unit of CMD) was determined as 75% by potentiometric titration method. 2.3. Preparation and characterization of blank nanoparticles and Rubone-loaded nanoparticles Emulsion-solvent evaporation method and electronic interactions were used to prepare blank CMD-PEI-PCL nanoparticles (BC-NPs) and Rubone-loaded CMD-PEI-PCL nanoparticles (RC-NPs). Briefly, 2 mL chloroform was used to dissolve PEI-PCL copolymer (20 mg) with or without a given amount of Rubone. The above solution was then mixed with 4 mL polyvinyl alcohol (PVA) aqueous solutions (1%). Subsequently, the above mixed solution was sonicated for



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5 min at 25 W in an ice bath using an ultrasonic cell disruption system. After vacuum evaporation, BP-NPs and RP-NPs were obtained by three times centrifugation at 8000 rpm for 15 min and washed three times with DI water. The precipitates were then collected and lyophilization to yield dried products. The Rubone concentration in the supernatant was measured in order to calculate the loading content (LC) and encapsulation efficacy (EE) of Rubone. High-performance liquid chromatography (HPLC) was used to detect the amount of Rubone. Water and acetonitrile 20:80 (v/v) was used as the mobile phase and the flow rate was controlled at 1 mL/min. Rubone was detected with UV detection at 399 nm. The LC and EE of Rubone were calculated as the following formula: LC (%) = (initial weight of Rubone - weight of Rubone detected in supernatant) / (weight of obtained RP-NPs) × 100% EE (%) = (initial weight of Rubone - weight of Rubone detected in supernatant) / (initial weight of Rubone) × 100% Subsequently, the coating of CMD over the formulated BP-NPs and RP-NPs was carried out through electronic interactions according to previous report.43 In brief, CMD was suspended in PBS 7.4 at a concentration of 10 mg/mL at constant stirring. The prepared BP-NPs or RP-NPs (1 mg/mL) were slowly added into the CMD solution. The reaction mixture was further stirred constantly at room temperature for 1 hour to ensure CMD adsorption. CMD-coated blank PEI-PCL nanoparticles (BC-NPs) and Rubone-loaded PEI-PCL nanoparticle (RC-NPs) were then obtained by centrifugation at 12, 000 rpm for 10 min. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) were used to analysis the particle size and zeta potential of the BP-NPs, RP-NPs, BC-NPs and RC-NPs (Malvern Instruments Ltd, Malvern, UK). All experiments were conducted at 633 nm and a constant angle of 90°. The morphology of nanoparticles was performed by transmission electron microscopy (TEM) measurement (EM-200CX, JEOL Ltd., Tokyo, Japan). 2.4. Fluorescence labeling of nanoparticles For cellular uptake and in vivo imaging studies, fluorescent dye Cyanine 3 monosuccinimidyl ester (Cy-3 NHS ester) was used for nanoparticle labeling. Cy-3-NHS ester group was conjugated with NH2 in PEI-PCL. Briefly, 1 mg of RP-NPs was dissolved in 5 mL of DI water, and 200 µg of Cy-3 NHS ester dissolved in 200 µL DMSO was added dropwise. The process was kept under


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nitrogen atmosphere, dark environment and under stirring at ambient temperature overnight, and finally centrifuged to obtain Cy-3 labeled RP-NPs and then washed with DI water to remove any residual reagent. And then the procedure of coating CMD onto Cy-3 labeled RP-NPs was the same as section 2.3. 2.5. In vitro Rubone Release, colloidal stability and buffering capability of nanoparticles Next, we used dialysis method to elucidate Rubone release behaviour. 5 mL of the RC-NPs (Rubone concentration, 160 µg/mL) was transferred into a dialysis bag (3500 Da, Millipore, USA), and dialyzed against 50 mL PBS of pH 7.4 or pH 5.2 with 10 % acetonitrile). PBS/Acetonitrile mixed solution instead of PBS only used in our experiment was to dissolve Rubone well. The release experiment was carried out at 37 °C water bath and vibrated at 100 rpm. 1 mL of medium was taken out at different time points, and analyzed for the released Rubone with HPLC as described above. After sampling, 1 mL of fresh PBS was added again. In analysing Rubone release profiles, the released Rubone was calculated and plotted at different time points. All of the experiments were carried out in three times. For evaluation of colloidal stability of various nanoparticles, BP-NPs, BC-NPs, RP-NPs and RC-NPs were suspended in 1 mg/mL BSA solution at 37 °C. The photographs were taken at 1, 3, 5, 10, 20 and 30 days to demonstrate the aggregation tendency of different nanoparticles formulations. The RC-NPs size was also determined at indicated time points by dynamic light scattering (DLS) analysis. Next, acid-base titration experiment was used to test the buffering capacities of the BC-NPs and BP-NPs. Briefly, BP-NPs and BC-NPs (0.5 mg/mL) were dissolved in 5 mL of 150 mM sodium chloride aqueous solution, respectively. The pH value of the solutions was then adjusted to 2.0 via adding 1 M hydrochloric acid. After adding 50 µL 0.1 M sodium hydroxide, the solution pH value was measured with a pH meter. Then, the obtained pH value was plotted. 2.6. Experimental cells and animals Human hepatocellular carcinoma cell line HepG-2 cells were cultured in DMEM medium containing 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin in a 5% CO2 incubator at 37 °C under 95% humidity. Healthy female Balb/c mice were obtained from Charles River Laboratories (Beijing, China). Animal feeding environment and all animal experiments were



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conducted in accordance with the local and ethical guidelines, and were approved by the Experimental Animal Ethics Committee in Beijing. 2.7. In vitro cellular uptake HepG-2 cells were cultured in DMEM media containing 10% FBS and incubated at 37°C incubator with 5% CO2. Confocal laser scan microscopy (CLSM) was used to assess the cell uptake and intracellular distribution of Cy-3-labeled RC-NPs. HepG-2 cells were seeded at a density of 1 × 105 cells per well at 37 °C in 2 mL DMEM medium and cultured overnight. Then, Cy-3-labeled RC-NPs were added into the medium. After 6 h incubation, the cells were washed twice with pH 7.4 PBS. Then, the cells were fixed using 4% formaldehyde. Cell nuclei were stained with DAPI and endosomes/lysosomes were stained with LysoTracker Green, respectively. Red fluorescence of Cy-3 and blue fluorescence of DAPI were observed using a Zeiss LSM780 confocal microscopy (Zeiss Co., Germany). 2.8. Cell proliferation assay HepG-2 cells were seeded in 96-well plates at a density of 1×104 cells/well and incubated at 37 °C incubator with 5% CO2. After incubation overnight, the culture media were replaced by 200 µL of fresh medium containing free Rubone dissolved in DMSO, BC-NPs, and RC-NPs for 48 h. Then, CCK-8 assay was used to assess cell viability at designed time points. The absorbance at 399 nm was measured using TECAN Infinite M200 microplate reader (Tecan, Durham, USA). Cell viability was calculated as following: Cell viability (%) = (absorbance of treated groupabsorbance of medium) / (absorbance of control group - absorbance of medium) × 100%. All experiments were conducted in three times. 2.9. Cell apoptosis assay AnnexinV/propidium iodide (PI) apoptosis detection kit (BD, USA) was used for apoptosis assessment. Cells treated with free Rubone and nanoparticles in different formulations containing the equivalent Rubone concentration of 20 µM were assessed. After 48 h, cells were trypsinized, collected and washed three times with PBS. Then, the cells were measured by flow cytometry (FACSCalibur, BD, USA) to determine the apoptosis ratio using the AnnexinV/propidium iodide apoptosis detection kit apoptosis detection kit. 2.10. In vitro cell migration assay


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HepG-2 cells were seeded in 6-well plates at a density of 6 × 105 cells/well. When the cell confluence reached 80%, the cells were scratched with a 1 mL sterile pipette tip. Different formulations were added into the media at the concentration of 20 µM of Rubone. 16 h incubation was allowed to recover the wound. Finally, the wound width in different groups was observed under a microscope, and quantified to evaluate the migration ratio. 2.11. Quantitative real-time PCR (qRT-PCR) In order to analyse the expression of mature miR-34a and analyse the gene expression of miR-4a target gene including Bcl-2, CDK6 and cyclin D1, qRT-PCR was used with internal standards as described previously.44 Briefly, Trizol reagent was used to extract total RNA. For measuring miR-34a expression, 1 µg of total RNA was transcribed into cDNA using Taqman MicroRNA Reverse Transcription Kit (Applied Biosystems). QRT-PCR measurement was performed using Taqman® Universal Master Mix II (Invitrogen, USA) and specific RT-primer designed for miR-34a. MiR-34a was normalized to an endogenous control U6. Then, standard PCR parameters were set as described previously.44 The primer sequences for miR-34a and U6 is 5’-AACAACCAGCTAAGACACTGCCA-3’

and

5’-GCTTCGGCAGCACATATACTAAAAT-3’, respectively. For measuring the miR-34a target genes expression, 1 µg of total RNA was transcribed using ImProm-IITM reverse transcription system (Promegra, USA) for first-strand cDNA. Gene expression was then detected by using the Brilliant® II SYBR® green qPCR master mix (Stratagene, USA). Then, standard PCR parameters were set as described previously.44 2.12. Western blot assay The cells treated with or without different Rubone formulations after 48 h incubation were harvested and lyzed with RIPA lysis buffer for 10 min on ice. After centrifugation, the supernatants were collected and the total proteins were quantified by Bradford’s reagent (Bio-Rad laboratories, USA). Total protein was subjected to 10% SDS-PAGE gel for electrophoresis, blocked for 1 h and transferred onto PVDF membranes and then incubated with the rabbit anti-Bcl-2, anti-CDK6 and anti-cyclin D1 antibody (Abcam, USA) at 4 °C overnight. After washing, the membranes were then incubated with the fluorescent dye-conjugated secondary goat anti-mouse antibody for 45 min. Finally, membranes were imaged with the Odyssey infrared



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imaging system (Li-COR Biosciences, USA) and quantified according to the instructions. GAPDH was used as an internal protein. 2.13. Hemolysis assay Hemolytic effects of BC-NPs were measured according to our previous methods.45 PBS and water was used as negative and positive controls, respectively. Different final concentrations of BC-NPs (1, 10, 50, 100, 200, 500 and 100 µg/mL) were used to assess the haemolytic activity of BC-NPs. The percentage of hemolysis was calculated using the following formula: Hemolysis % = (sample absorbance-negative)/positive control-negative) × 100% 2.14. In vivo imaging and biodistribution analysis In vivo images of nude mice after intravenous injection of Cy-3-labeled RC-NPs were carried out by the ex/in vivo imaging system (CRi, Woburn, MA) to collect the fluorescent signals of Cy-3 at 1, 6, 12, and 24 h after tail vein injection with 100 µg of Cy-3-labeled RC-NPs. The mice were sacrificed after in vivo imaging and the tumors and other major organs were collected for imaging. 2.15. In vivo anti-tumor activity of RC-NPs Female nude Balb/c mice (4-6 weeks) were obtained from Charles River Laboratories (Beijing, China) and housed in a pathogen-free condition. A human HCC xenograft tumor model was established by subcutaneously injection of HepG-2 cells (5 × 106) in the right axilla. The mice were randomly divided to five groups with five mice in each group when the tumors volumes reached around 100 mm3. The mice were then intravenous injected with saline, BC-NPs and RC-NPs (Rubone of 10 mg/kg), respectively. Total five injections were performed every 3 days. The body weights were measured and tumor volumes were measured and calculated every 3 days according to the following formula: Tumor volume = (length × width2)/2. The animals were then sacrificed, and tumor tissues and major organs were collected for further analysis. 2.16. Tumor and major organ histology The collected tumors and major organs were fixed in 10% formalin, embedded into paraffin, cut at 4 µm thickness, and then stained with hematoxylin and eosin (H&E) by the Peking University Health Science Center (PUHSC). The tissue sections were then examined by microscopy and analyzed by an experienced veterinary pathologist. 2.17. Statistical analysis


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Each experiment was performed at least three times. Mean ± SD was determined for all data. Statistically significant differences (*p

Carboxymethyl Dextran-Stabilized Polyethylenimine-Poly(epsilon

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