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Hyaluronic acid engineered nano-micelles loaded with 3, 4-difluorobenzylidene curcumin for targeted killing of CD44+ stem-like pancreatic cancer cells PRASHANT KESHARWANI, Sanjeev Banerjee, Subhash Padhye, Fazlul H Sarkar, and Arun K Iyer Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00941 • Publication Date (Web): 24 Aug 2015 Downloaded from http://pubs.acs.org on August 27, 2015
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Hyaluronic acid engineered nano-micelles loaded with 3, 4difluorobenzylidene curcumin for targeted killing of CD44+ stem-like pancreatic cancer cells
Prashant Kesharwani, PhDa, Sanjeev Banerjee, PhDb, Subhash Padhye PhDc, Fazlul H. Sarkar PhDb, Arun K. Iyer PhDa,d,*
a Use-inspired Biomaterials & Integrated Nano Delivery (U-BiND) Systems Laboratory, Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, 259 Mack Ave, Wayne State University, Detroit, MI 48201, USA b Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, 740 HWCRC, Detroit, Michigan 48201, USA c Interdisciplinary Science & Technology Research Academy, Department of Chemistry, Abeda Inamdar College, Azam Campus, University of Pune, Pune 411001, India d Molecular Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, Michigan, 48201, USA
*Corresponding author Arun K. Iyer, Ph.D. Department of Pharmaceutical Sciences Eugene Applebaum College of Pharmacy and Health Sciences 259 Mack Ave, Room 3601 Wayne State University, Detroit, MI 48201 Phone: 313-577-5875 Fax: 313-577-2033 Email:
[email protected] (A.K. Iyer) Disclosures: There is no conflict of interest and disclosures associated with the manuscript. 1
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ABSTRACT Cancer stem-like cells (CSLCs) play a pivotal role in acquiring multidrug resistant (MDR) phenotype. It has been established that pancreatic cancers overexpressing CD44 receptors (a target of hyaluronic acid; HA) is one of the major contributors for causing MDR. Therefore, targeted killing of CD44 expressing tumor cells using HA based active targeting strategies may be beneficial for eradicating MDR-pancreatic cancers. Here, we report the synthesis of a new HA conjugate of co-poly(styrene maleic acid) (HA-SMA) that could be engineered to form nanomicelles with a potent anticancer agent, 3, 4-difluorobenzylidene curcumin (CDF). The anticancer activity of CDF loaded nano-micelles against MiaPaCa-2 and AsPC-1 human pancreatic cancer cells revealed dose-dependent cell killing. Results of cellular internalization further confirmed better uptake of HA engineered nano-micelles in triple-marker positive (CD44+/CD133+/EpCAM+) pancreatic CSLCs compared with triple-marker negative (CD44/CD133-/EpCAM-) counterparts. More importantly, HA-SMA-CDF exhibited superior anticancer response towards CD44+ pancreatic CSLCs. Results further confirmed that triple-marker positive cells treated with HA-SMA-CDF caused significant reduction in CD44 expression and marked inhibition of NF-κB that in-turn can mitigate their proliferative and invasive behavior. Conclusively, these results suggest that the newly developed CD44 targeted nano-micelles may have great implications in treating pancreatic cancers including the more aggressive pancreatic CSLCs. KEYWORDS Pancreatic cancer; hyaluronic acid; copoly(styrene maleic acid); 3, 4Difluorobenzylidene curcumin;; cancer stem like cells; cancer stem cells; CD44 targeting; nanomicelles.
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INTRODUCTION Pancreatic cancer is one of the most deadly malignancies in the United States, with a five-year survival rate of less than 6 %
1,2
. Upon diagnosis, 80 % of pancreatic cancer cases are deemed
inoperable due to the high risk of tumor infiltration with surrounding blood vessels and digestive ducts
3–5
. Due to the complex nature of malignant pancreatic cancers the incidence and death
rates have been increasing although the overall cancer incidence and death rates are declining for other cancers 6. Additionally, more than half of the pancreatic cancer patients are diagnosed when they have advanced disease that has spread to other organs 6. Indeed, none of the current chemotherapy regimens provide more than one-year survival benefit for pancreatic cancers. These shortcomings of the conventional clinical therapies against aggressive and metastatic pancreatic cancers accentuate the urgent need for targeted therapies and delivery technologies for achieving better treatment outcome. Recent studies in mouse models of pancreatic ductal adenocarcinomas have revealed that cellular dissemination leading to metastasis occurs prior to the formation of identifiable primary tumor 7. This behavior is associated with epithelial-to-mesenchymal transition (EMT). It is also known that circulating pancreatic tumor cells maintain a mesenchymal phenotype and express CD44, a known feature of cancer stem cells (CSCs) 8. Evidence for the existence of CSCs or cancer stem like cells (CSLCs) has been provided earlier in primary human pancreatic adenocarcinomas grown in immuno compromised mice as well
9,10
. Furthermore, CD44 has
received considerable attention as a target receptor for pancreatic cancer therapy because it plays critical roles in CSC maintenance and resistance to chemo- and/or radiotherapy
11–13
. These
results clearly indicate that CD44 targeted therapy may be an useful approach for treating
3
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pancreatic cancers including the tumors with more aggressive features such as EMT, CSCs or CLSCs. For the current study, we selected styrene-maleic acid (SMA) co-polymer with a relatively low molecular weight of 1.6 kDa along with hyaluronic acid (HA) of 10 kDa as our starting building blocks based on their prior favorable properties
14–18
. HA is biodegradable,
biologically inert, non-toxic, non-immunogenic and non-inflammatory, which makes it an ideal carrier polymer for systemic drug delivery applications
19
. Also, a relatively simple coupling
chemistry of SMA anhydride allows for modification of the sugar residues on HA polymer to arrive at functional macrostructures that facilitates self-assembly and encapsulation of hydrophobic drugs
14,20
. Of primary importance to pancreatic tumor selective delivery, the HA
backbone in itself is endowed with tumor targeting moieties (Scheme 1) that specifically recognizes CD44, an integral membrane glycoprotein over-expressed on several tumors cell surfaces, including pancreatic cancers and cancer stem like cells (CSLCs). In terms of the chemotherapeutic agent, curcumin (CMN) has long been used in Indian Ayurvedic Medicine for treating many health disorders. Incidentally, a growing body of evidence demonstrates that CMN can prevent multiple cancers including pancreatic cancers. However, The therapeutic activity of CMN has been impeded by its very poor aqueous solubility, low bioavailability, rapid metabolism and clearance. Many researchers have attempted to deliver CMN using micelles, liposomes, and nanoparticles
21–24
. Notably, CMN nano-micelles showed
many fold improved half-life 25–27; however, CMN was found unstable once released from nanomicelles due to rapid metabolism and clearance. Due to these shortfalls, many groups have tried to synthesize CMN analogues that display better stability, increased bioavailability with similar or better biological activity as that of CMN
12,28
. Along these lines, we have successfully 4
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developed a novel 3, 4-difluorobenzylidene curcumin (CDF) derivative, which showed improved stability, higher therapeutic potential and 16-fold increased half-life compared to CMN. CDF also displayed exceptionally improved pancreas selective accumulation
28–30
. Very recently,
Basak et al. reported that the nude mice xenograft study showed a statistically significant tumor growth inhibition of UM-SCC-1R cells and a reduction in the expression of CD44, indicating promising inhibitory effect of liposomal CDF on CSCs 31. More prominently, we found that CDF could inhibit the growth of CSCs, and induce disintegration of colonospheres that are highly enriched in CSCs
32
. These findings clearly demonstrate the utility of CDF as a promising
therapeutic agent for treating pancreatic cancer-stem-like cells (CSLCs). However, poor aqueous solubility of CDF, although better than CMN, has made its systemic administration problematic. In this context, we have taken advantage of our previous findings that amphiphilic SMA could be used as a nano-micellar platform for fabricating self-assembling nano-micelles of CDF 33,15. In the current study, we have engineered CD44 targeting HA-SMA conjugate based selfassembling nano-micelles for targeted delivery of CDF, with the aim to treat human pancreatic cancers including CD44+ pancreatic CSLCs. It has been hypothesized that, after systemic administration, the developed nano-formulation (HA-SMA-CDF) would reach the tumor site based on the enhanced permeability and retention (EPR) effect
34–36
as well as internalize into
tumor cells over-expressing the target by active targeting mechanism specifically via CD44 receptor mediated endocytosis (please see graphical abstract) 16,37. EXPERIMENTAL SECTION Materials. CDF was synthesized as described earlier
28,38
. Poly(styrene-co-maleic
anhydride), SMA (average molecular weight~1.6 kDa), N-(3-Dimethylaminopropyl)-N’-
5
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ethylcarbodiimide
hydrochloride
(EDC)
and
3-[4,
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5
Dimethylthiazol-2-yl]-2,
5
Diphenyltetrazolium Bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO). HA (low molecular weight) (Average Mw 10 kDa) was purchased from Lifecore Biomedical (Chaska, MN). All other chemicals were of reagent grade and used without any modification. Synthesis of CD44 targeted HA-SMA conjugate (HA-SMA). For engineering the targeted nano-micelles, HA-SMA conjugate was first synthesized by adding known amounts of HA in NaHCO3 buffer with fixed amounts of anhydrous SMA to afford the anhydride ring opening reaction of SMA with the alcohol groups of HA. For this purpose, HA was dissolved in deionized (DI) water at room temperature (RT) and 1M NaHCO3 was added slowly to the HA solution and stirred for 1 h at RT. The pH was adjusted to 8.9 with 1M NaOH. In another beaker, styrene maleic anhydride polymer was dissolved in anhydrous DMSO and added drop wise to the alkaline HA solution under vigorous stirring. The reaction medium was stirred until the solution become clear. The so-formed HA-SMA conjugate was purified by ultrafiltration using Millipore tangential flow filtration (TFF), (Millipore, Milford, MA) followed by lyophilization (Scheme 2a) and characterization by proton nuclear magnetic resonance spectroscopy (1H-NMR) and Fourier transform infrared spectroscopy (FTIR). Fabrication and characterization of SMA-CDF and HA-SMA-CDF nano-micelles. SMA-CDF (non-targeted nano-micelles) and HA-SMA-CDF (targeted nano-micelles) were fabricated according to previously reported method
15,34,17,39,40
. In brief, known amount of the
newly synthesized HA-SMA conjugate or hydrolyzed amphiphilic styrene-maleic acid copolymer (SMA) was dissolved separately in DI water at RT under magnetic stirring and the pH was adjusted to 5.0. CDF was dissolved in minimum quantity of DMSO and added drop wise to polymer solution. The instantaneous self-assembly of the styrene component of SMA and 6
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hydrophobic domains in CDF resulted in the formation of nano-micelles. Subsequently, EDC was added drop wise and stirred for 30 minutes in the dark to minimize CDF degradation due to light exposure. The pH was maintained at 5.0 throughout this process. Following this, the soformed nano-micelles were precipitated by 1M HCl and centrifuged at 5000 rpm for 15 min. The precipitated material was collected and re-dissolved in DI water and the pH was raised to 10.0 by drop wise addition of 1M NaOH until the suspension become clear. Finally, the pH was readjusted to 7.4 using 0.1M HCl and dialyzed extensively overnight using dialysis membrane (molecular weight cut-off 8 kDa, Spectrapor, Spectrum Labs, SD) against distilled water to remove un-encapsulated free drug (CDF). Finally the SMA-CDF and HA-SMA-CDF nanomicelles were lyophilized (Eyela Inc., Tokyo, Japan) (Scheme 2b). Characterization of nano-micelles. Nano-micelles were characterized for size and zeta potential using a Beckman Coulter Delsa Nano C DLS Particle analyzer (Beckman Coulter, Inc., Fullerton, CA) equipped with a 658 nm He-Ne laser as reported by us earlier
40
. In addition,
nano-micelles were further characterized for surface morphology by transmission electron microscopy (TEM) and atomic force microscopy (AFM) as reported by us earlier 40. Drug loading in nano-micelles and in vitro release studies. To make sure that the CDF was chemically unaltered during micelle formation, SMA-CDF and HA-SMA-CDF nanomicelles were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using Waters 2695 separations module and LC system coupled with a Waters Quattro Micro™ triple quadrupole mass-spectrometric detector equipped with an electrospray ionization source (Milford, MA, USA). CDF was monitored in negative ionization mode at the transition of m/z, 367.1→148.8 and 491.1→216.9, respectively. The internal standard zileution was monitored in the positive mode at the transition of m/z, 237.1→160.8. Results for free CDF and nano-micelles 7
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were compared to identify changes, if any. Further, the amount of drug loading in the nanomicelles was also determined by LC-MS/MS method. For this purpose, firstly the stock solution was prepared by dissolving desired concentration of CDF or CDF nano-micelles followed by vortex mixing for 10 min and centrifugation at 10000 rpm for 15 min twice. The supernatant was transferred to a new centrifuge tube each time. The final supernatant was diluted with 70:30 MeOH : water to desired concentration, and final concentration 0.2 µg/ml Zileuton was also added as internal standard 41. The nano-micelles were subjected to in vitro drug release studies using dialysis membrane diffusion technique in PBS (pH 5.5, 7.4 and 10.0) against DI water as receptor medium. Ten milligrams of non-targeted and targeted nano-micelles was separately placed inside the dialysis tubing (3.5 KDa, Sigma, USA), hermetically sealed and suspended immediately in 50 ml of released media (buffer pH 5.5, 7.4 and 10.0) under sink conditions. The volume of receptor compartment was maintained constant by replenishing it with 1 ml of sink solution, after withdrawing of 1 ml aliquot. The sample was analyzed by UV spectroscopy to determine the CDF content. This experiment was repeated thrice and the results are presented as mean ± SD (n=3). Stability study of nano-micelles under storage condition. Stability studies were performed to validate the developed formulation in terms of physical change/stability and drug leakage. For this purpose, the non-targeted and targeted nano-formulations were stored in the dark in amber colored vials and in colorless glass vials at (a) 0°C; (b) at ambient temperature ranging from 20–30°C; and (c) at 60±2°C in controlled ovens for a period of 6 weeks and examined every week for drug leakage. Drug leakage was determined by monitoring the release
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of drug from the formulation after storage at different temperature by UV spectrometry (Jasco 530 UV-Visible spectrometer, Tokyo, Japan). In order to evaluate the hemocompatibility and serum stability, the fabricated nanomicelles (1 mg/mL prepared in PBS) was incubated in the presence of fibrinogen (1 mg/mL in PBS 7.4) or 50 % FBS (prepared in 1X PBS, pH 7.4) at 37°C. Any change in size of the nanomicelles was assessed by DLS at predetermined time points up to 4 days and was used as a parameter to evaluate the stability of nano-micelles (n=3) 40. Cell culture. Human pancreatic cancer cell lines MiaPaCa-2 and AsPC-1 were used for our study on the basis of their sensitivities to CDF, as reported earlier
42
. Both cell lines were
maintained in Dulbecco's Modified Eagle's Medium (DMEM; Fisher Scientific, Waltham MA), supplemented with 5% FBS (Fisher Scientific, Waltham MA), 2 mmol/L glutamine, 50 units/mL penicillin, and 50 µg/mL streptomycin as standard culture condition 42. The cell lines have been tested and authenticated by the core facility of Applied Genomics Technology Center at Wayne State University. The method used for testing was short tandem repeat profiling using the PowerPlex 16 System from Promega (Fitchburg, WI) 43. The CSLCs (triple-marker positive; CD44+/CD133+/EpCAM+) and triple-markernegative (CD44-/CD133-/EpCAM-) cells were isolated from human pancreatic MiaPaCa-2 cells, by fluorescence-activated cell sorting (FACS) technique. The post-sorted cells were subsequently maintained in 5% FBS–DMEM media at 37°C under standard culture condition 44. In vitro cancer cell viability assay. In short, cells were seeded in a 96-well culture plate and the control and test formulations were added 24 h after seeding as freshly prepared solutions in PBS (pH 7.4) between 100 to 1000 nM concentrations. MTT (0.5 mg/ml) was added at the end 9
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of treatment (72 h) and plates further incubated at 37°C for 2 h, followed by replacement of media with DMSO at RT in a shaker for 30 min. The absorbance was measured at 595 nm using Ultra multi-functional micro plate reader (TECAN, Switzerland) and percent cell viability was determined by comparing the results with appropriate controls 45. Quantitative cellular uptake studies of CDF loaded nano-micelles in pancreatic cancer cells. CDF loaded targeted (HA-SMA-CDF) and non-targeted (SMA-CDF) nano-micelle formulations were used for cellular uptake studies. MiaPaCa-2 cells (5000 cells per well) were seeded in a 96-well culture plate and allowed to attach for 24 h. The medium in the well was replaced with varying concentrations of free CDF (control) or CDF loaded nano-micelles and incubated for different periods of time (0.5-4h) at 37°C in a CO2 incubator. At the end of the incubation, the media was removed from the wells and the cells were rinsed thrice with ice cold PBS to remove the unbound nanoparticles outside the cells. Subsequently, 100 µl of 0.1% Triton X-100 in 0.1 N NaOH solutions was added to lyse the cells. Chloroform (0.5 ml) was added to the cell lysates, homogenized for 1 min (Polytron, Kinematica Inc., Switzerland) and centrifuged at 8000 rpm for 15 min (Thermo Electron Corp, Waltham, MA). The organic extract was separated and used to measure the absorbance of CDF at 447 nm (λmax of CDF) using a UV spectrometer (Jasco 530 UV-Visible spectrometer, Tokyo, Japan). Anticancer activity of targeted and non-targeted nano-micelles on CD44+ Versus CD44-pancreatic cancer cells. Triple-marker positive (CD44+/CD133+/EpCAM+) CLSCs and triple-marker-negative (CD44-/CD133-/EpCAM-) cells were isolated and cultured as described in the methods section and seeded in a 96-well culture plate, and subsequently free CDF, HASMA, non-targeted (SMA-CDF) and targeted (HA-SMA-CDF) nano-micelles were applied 24 h after seeding as freshly prepared solution in DMSO diluted with PBS (for CDF) or PBS (for the 10
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nano-formulations) (pH 7.4) between 250 to 750 nM concentration. MTT reagent was added and the absorbance was measured at 595 nm using Ultra Multi-functional Micro plate Reader (TECAN, Switzerland) and percent cell viability was determined by comparing the values with appropriate controls. Effect of CDF nano-formulation on the expression of CD44 by western blot analysis. Triple marker positive CSLCs derived from MiaPaCa-2 were plated and allowed to attach for 36 h. CDF loaded formulations were directly added to cell cultures at 250 nM concentration and incubated for 48 h. Control cells were incubated in the medium containing an equivalent concentration of PBS. After incubation, the cells were collected in PBS and whole cell lysate was prepared by suspending the cells in 150 µL of lysis buffer [1 mol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% Triton X-100; 0.1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 2 µg/mL leupeptin, 2 µg/mL aprotinin], lysed and centrifuged. The cells were disrupted by sonication (Branson Ultrasonics, Danbury, CT) and the total protein content was determined by bicinchoninic acid (BCA) assay. For immunoblotting, 30 µg total proteins was separated on SDS-PAGE, electro-transferred onto nitrocellulose membranes, and probed with specific antibody (CD44 signaling, MA). Detection of specific proteins was carried out with an enhanced chemiluminescence Western blotting kit according to manufacturer’s instructions (Life Technologies, Waltham, MA). Electrophoretic mobility shift assay (EMSA). To investigate the effect of targeted nano-micelles
(HA-SMA-CDF)
in
down
regulating
NF-κB
activity;
we
performed
electrophoretic mobility shift assay (EMSA) and measured its activity on NF-κB DNA-binding protein label using extracts from CSLCs+ and CSLCs- MiaPaCa-2 cells treated with HA-SMACDF (250 nM concentration; 24 h). Nuclear extracts were prepared from treated samples, and 11
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EMSA was performed by incubating 10 µg of nuclear protein extract with IR Dye TM–700 labeled NF-κB oligonucleotide according to the method reported by us earlier
46
. The DNA-
protein complex formed was visualized by Odyssey Infrared Imaging System (Odyssey CLx, LICOR Biosciences, Lincoln, NE) using Odyssey Software Release 1.1). Statistics. The statistical analysis of data was performed using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. The results are expressed as mean ± standard deviation and n showing the number of repeats. A difference of p
