LipidâPolymer Nanoparticles Encapsulating Doxorubicin and 2

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Lipid-polymer nanoparticles encapsulating doxorubicin and 2’-deoxy-5azacytidine enhance the sensitivity of the cancer cell to chemical therapeutic Xianwei Su, Zhaohui Wang, Lili Li, Mingbin Zheng, Cuifang Zheng, Ping Gong, Pengfei Zhao, Yifan Ma, Qian Tao, and Lintao Cai Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300675c • Publication Date (Web): 09 Apr 2013 Downloaded from http://pubs.acs.org on April 14, 2013

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Molecular Pharmaceutics

Lipid-polymer nanoparticles encapsulating doxorubicin and 2’-deoxy-5-azacytidine enhance the sensitivity of the cancer cell to chemical therapeutic

Xianwei Sua,1, Zhaohui Wanga,1, Lili Lia,c, Mingbin Zhenga,b, Cuifang Zhenga, Ping Gonga, Pengfei Zhaoa, Yifan Maa, Qian Taoa,c,*, Lintao Caia,*

a

Guangdong Key Laboratory of Nanomedicine, Shenzhen Key Laboratory of Cancer Nanotechnology,

Institute of Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China b

Department of Chemistry, Guangdong Medical college，Dongguan 523808, PR China

c

Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and

Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong

Keywords: Drug delivery; chemotherapeutic drug; epigenetic drug; cancer therapy; combined therapy

1

These authors contributed equally to this work. To whom correspondence may be addressed. E-mail address: [email protected] (Lintao Cai), [email protected] (Qian Tao); *

1

ACS Paragon Plus Environment


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Abstract

Synergistically repress cancer cell growth PLGA Doxorubicin Epigenetic drug-DAC Soybean lecithin DSPE-PEG-COOH

Nanomedcine holds great potential in cancer therapy due to its flexibility on drug delivery, protection, releasing, and targeting. Epigenetic drugs, such as 2’-deoxy-5-azacytidine (DAC), are able to reactive expression of tumor suppressor genes (TSG) in human cancers, therefore, might be able to enhance the sensitivity of cancer cells to chemotherapy. In this report, we fabricated a lipid-polymer nanoparticle for co-delivery of epigenetic drug DAC and traditional chemotherapeutic drug (DOX) to cancer cells, and monitored the growth inhibition of the hybrid nanoparticles (NPs) on cancer cells. Our results showed that NPs encapsulating DAC, DOX, or both, could be effectively internalized by cancer cells. More importantly, incorporating DAC into NPs significantly enhanced the sensitivity of cancer cells to DOX by inhibiting cell growth rate and inducing cell apoptosis. Further evidence indicated that DAC encapsulated by NPs was able to rescue the expression of silenced TSG in cancer cells. Overall our work clearly suggested that the resulting lipid-polymer nanoparticle is a potential tool for combining epigenetic therapy and chemotherapy.

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Introduction

Effectiveness of traditional chemotherapeutic drugs is greatly limited by nonspecific targeting and accumulated toxicity in health organs, where the therapeutic molecules not only kill the cancer cells but also the normal cells. Thus, the therapeutic efficacy to cancer tissue is dramatically reduced, while the accumulated drug in normal organs may lead to organ defects. For example, cumulative doxorubicin (DOX) administration (over 550 mg/m2) may induce severe cardiac side effects, including congestive heart failure, dilated cardiomyopathy, and death 1. Furthermore, the instability of chemotherapeutic agents in opening to circulation system or the nonspecific binding to serum proteins may add another barrier for delivering drugs to targeting tumor tissue.

Nanoparticle (NP) based drug delivery systems for cancer therapy developed rapidly in recent years

2-4

.

Compared to traditional chemotherapeutics, nanoparticles have several advantages: (1) NPs may increase the tumor specific distribution through the enhanced permeability and retention effect (EPR) 5-6; (2) by conjugated with specific tumor cell targeting ligands, such as antibody, aptamers, folic acids, the specificity of NPs on tumor cell targeting may be further increased 7; (3) NPs can load large amount of drugs, protect them from the circulation cleaning system thus prolong the half life of anticancer drugs in bloodstream, and increase the effective drug concentration at the targeting site. In addition, NPs can encapsulate multiple types of drugs or agents with different anti-cancer mechanisms, therefore to acquire synergetic anti-cancer effects. For example, a PEG-lipid nanoparticle was used to co-deliver an anti-angiogenesis agent combretastatin and a pro-apoptosis agent doxorubicin 8. This combined treatment showed significant synergetic suppression on the tumor growth.

Importantly, NPs are potential tools in overcoming the frequent developed multidrug resistance (MDR) in chemotherapeutics. In many cases, traditional chemotherapeutics were effective in the beginning of treatment, but the patients ended up with drug resistant to tumor progression or recurrence. There are multiple mechanisms contribute to the MDR 7,9-11, such as loss sensitive to apoptosis, activation of DNA repair, and increased drug efflux via the ATP-binding cassette (ABC) superfamily. Many NPs based 3



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strategies aimed at inhibiting MDR had been reported. For example, delivery of inhibitors or siRNAs to drug efflux pump MDR1/P-gp by NPs had been extensively studied by many groups

12-15

. In another

instance, codelivery of DOX and an siRNA targeting the anti-apoptosis protein BCL-2, increased the cytotoxic activity of DOX dramatically (132 folds) 16.

One of the important strategies to regulate drug sensitivity is to modulate the epigenetic status of tumor suppressor genes (TSGs) or drug resistance related genes. Silence of TSGs by epigenetic modification such as DNA promoter methylation and histone deacetylation mediated chromatin remodeling play important roles in every stage of tumor progression

17-18

. Remarkably, this epigenetic induced silencing

can be reversed by inhibitors to DNA methyltransferase like 5-azacytidine and 2’-deoxy-5-azacytidine (DAC) or to histone deacetylase (HDAC) like Trichostatin A (TSA) 19-20. Therefore these inhibitors serve as potential anti-cancer drugs, and some of them had been approved in clinical usage 21. Compared to regular chemotherapeutic drugs, these inhibitors have much less cytotoxicities; and are able to reactive multiple TSGs in different pathways, such as the key regulators on RAS pathway, hMLH1 in mismatch DNA repair, caspase 8 in apoptosis

10-11,22-25

. It has been demonstrated that combining the epigenetic

inhibitors with traditional chemotherapeutics could effectively increase the cellular drug sensitivity 26-29. For example, in one of these tests, usage of DAC had successfully reversed the drug resistance to cisplatin in A2780/cp70 cell line 26.

Herein, we reported the co-delivery of epigenetic drug DAC and traditional chemotherapeutic drug DOX using lipid-polymer based PLGA-lecithin-PEG nanoparticle. By examining the growth repression effects on cultured cancer cells, such as growth rate inhibition and apoptosis promotion, our results clearly showed that nanoparticle encapsulated epigenetic drug DAC synergistically promoted the cell-growth-inhibition effect of DOX. Therefore may provide a powerful tool for the future anti-MDR cancer therapy.

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Materials and Methods Chemicals PLGA (D,L-lactide-co-glycolide, MW: 5000-15000, lactide: glycolide (50:50)), 2’-deoxy-5-azacytidine (DAC), Hoechst 33258, were purchased from Sigma-Aldrich Chemical Co. Soybean lecithin, DSPE-PEG-COOH (1,2-distearoyl-sn- glycero-3-phosphoetha-nolamine-N-[carboxy (polyethylene glycol) 2000]) were purchased from Avanti Polar Lipids, Inc. Doxorubicin (DOX) was purchased from Jinhe Bio-Technology Co. Ltd. 3,3’-Dioctadecloxacarbocyanineperchlorate (Dio) was purchased from Life Technologies Co. Deionized water was produced by Milli-Q Plus System (Millipore Co.). All other chemicals were of analytical grade and used as received.

Assembly of Lipid-polymer Nanoparticles PLGA was dissolved in acetonitrile solution at concentration of 2 mg/ml. Soybean lecithin and DSPE-PEG-COOH were dissolved in chloroform:methanol solution (v/v = 9:1) at concentration of 1 mg/ml and 25 mg/ml respectively, and then mixed at a molar ratio of 6:4 and dissolved in 4% ethanol aqueous solution. DOX and DAC were freshly prepared in water solution at 1 mg/ml and 1 mg/ml respectively. The drug weight compare to the PLGA was about 10%; the total lipid weight (lecithin +DSPE–PEG–COOH) was 20% of the PLGA polymer. The lipid mixture solution was preheated at 65°C for 3 min, and the PLGA solution encapsulating chemotherapeutic and epigenetic drug was added under gentle stirring dropwisely. The mixed solution was vortexed vigorously for 3 min followed by gentle stirring for 4 hrs at 65°C. Finally the nanoparticles were washed three times by ddH2O, using an Amicon ultra-4 centrifugal filter (Millipore Co.) with a molecular weight cutoff of 10 kD at 7500g, 15 min to remove the free drug molecules. At last the nanoparticles were dissolved in 1xPhosphate Buffered Saline (PBS, pH=7.4) (as shown in Fig. 1A). After the drugs were released in 90% DMSO, the concentration of DAC encapsulated in the NPs could be determined by Lamda UV spectrophotometer (SPECTRO Analytical Instruments) at 254 nm absorption, and the doxorubicin concentration was determined by the Fluorescence spectrometer at 480 nm absorption. The same procedures were used to prepare the nanoparticles encapsulated either single drug, or combined two drugs (DOC+DAC), or in combination with additional Dio fluorescent dye (Dio was premixed with lecithin+DSPE-PEG-COOH lipid mixture 5



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solution at a final concentration of 300 µg/ml).

Characterization of the lipid–polymer hybrid NPs After resuspended in PBS, the hydrodynamic diameters (nm) of the nanoparticles and their polydispersity indexes were determined from three repeated experiments using dynamic light scattering with a Zeta Pals instrument (Brookhaven Instruments) at 25°C. The Zeta potentials (mV) of the nanoparticles were calculated from three repeated measurements on a DelsaTM Nano C (Beckman Instruments). The morphology of NPs was observed by transmission electron microscopy (TEM, JEM-2100, JEOL Ltd) using the negative staining method as described before 30.

HPLC chromatographic conditions HPLC analyses of the drugs or NPs were performed on SHIMADZU LC-20A HPLC system with the Inertsil® ODS-SP 4.6x250 mm column (SHIMADZU Co.). For DAC analyses, DAC solution or NPs were diluted in 10 mM pH 6.8 potassium phosphate buffer containing 30% DMSO and 50% acetonitrile. The DAC was eluted at flow rate of 0.75 ml/min with 10 mM pH6.8 potassium phosphate buffer as the mobile phase 31. The amount of DAC was quantified by the absorbance of 254 nm. For DOX analyses, DOX solution or NPs were diluted in DMSO:H2O (80:20) solution, and eluted at flow rate of 0.75 ml/min with pH3.0 methanol:H2O (60:40) solution (containing 0.1% ammonia solution (25%), and adjusted to pH3.0 with formic acid) as mobile phase

32

. The amount of DOX was quantified by

fluorescent detection with 475 nm and 580 nm as excitation and emission wavelengths respectively.

Drug release assay for lipid–polymer hybrid NPs in PBS After preparation, the NPs were stored at 4°C. At day 5, 10, 15 and 20, 100 µl NPs were spun by using the Amicon ultra-4 centrifugal filters (cutoff MW 10kD) at 7500g, 15 min. Then collected the flow-through supernatants and applied on HPLC quantitative analyses. The amounts of DOX in the free form were then normalized to the total amount of DOX in NPs-DOX-DAC solution in 0 day to indicate the percentages of drug releasing. Three individual repeats were done for each sample.

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Cell culture The MDA-MB231 (MB231), HONE1 cell lines, obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were grown in RPMI 1640 (Hyclone, Thermo-Fisher Scientific Inc.) containing 10% fetal bovine serum (FBS, Hyclone), 2 mM l-glutamine, Penicillin-streptomycin solution (40 U/ml each, Gibico, Life Technologies Co.) at 37°C in a humidified CO2 (5%) incubator.

Cell viability assay The

cytotoxicities

of

the

nanoparticles

were

evaluated

by

MTS

([3-(4,5-dimethylthiazol

-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt;) assay (Promega Co.). Cells were seeded (2×103 cells/well) in 96-well microtitre plates. After 24 hrs incubation, the growth media were replaced with 100 µl fresh RPMI 1640 media containing 10% FBS and different concentration of nanoparticles encapsulating DOX (0-2 µg/ml), or DAC (0-200 µM), or the combined two drugs respectively. Cell proliferation were then examined by the absorbance of 490 nm every 24 hrs for another 5 days. Before MTS assay, the medium was replaced with 100 µl fresh growth medium, then added 20 µl MTS and incubated for 4 hrs. Thereafter, absorbance was measured at 490 nm (reference wavelength 630–700 nm may be used) using a microplate reader (Synergy4, BioTek Instruments Inc.). The cell viability (%) was calculated according to the following equation: ODsample − ODblank

Cell viability (%) = ODcontrol − ODblank ODsample, ODcontrol represented the absorption from the cells treated with nanoparticles and PBS respectively, and ODblank represented the absorption from medium only. For each time point, absorbance at 490 nm from three individual culture wells was measured.

Cellular uptake assay for lipid–polymer hybrid NPs MB231 cells were seeded in 35 mm glass bottom cell culture dishes (2x105 cells/dish), and further incubated for 24 hrs. The cells were treated with nanoparticles for 2 hrs. After incubation, the cells were washed twice with PBS, then fixed by 4% PFA for 30 min at room temperature, and washed by PBS for another three times. Hoechst 33258 (1000x) was added to stain the nuclei for 10 min, followed by three 7



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times washing and a final resuspension with PBS (pH 7.4). The fluorescent images were observed under Leica TCS SPS confocal microscope system.

To quantitative measure the uptake of NPs, HONE1 and MB231 cells were seeded in 24 well/plate (1x105 cells/well). After incubated for 24 hrs, the cultures were changed with fresh media, and added DOX solution or NPs-DOX to the DOX final concentration of 0.2 µg/ml. At different time points, collected the culture media for quantitative HPLC analyses as described above. Three individual elution cycles were performed for each sample. At the same time point, the cells were harvested, washed once with PBS, resuspended in PBS; and the fluorescence of DOX was analyzed using Accuri® C6 Flow Cytometer (BD Biosciences).

Apoptosis assay of nanoparticles effect on cancer cell line Cells were freshly seeded at a density of 1x105 cells/well in 6 wells plate. After overnight incubation, cells were treated with nanoparticles encapsulating DOX, DAC or combined two drugs for 3 days. The cells were harvested and treated following the instruction of the Annexin V FITC Apoptosis Detection Kit (Merk Co.). The fluorescence was measured using FACS Calibur system (BD Biosciences). All experiments were performed in triplicate.

Restoration of Tumor suppression gene (TSG) expression Cells were seeded at a density of 1x105 cells/well in 6 wells plate for 24 hrs. Then replaced the medium with fresh medium containing 5 µM DAC, and changed the medium every 24 hrs. Or the cells were treated with DAC or NPs-DAC (DAC final concentration to 5 µM) for continuous 72 hrs. Cells treated with NPs without drug for 72 hrs were used as blank control. Total cellular RNA was purified from cells using the Trizol reagent (Life Technologies Co) according to the manufacturer’s instructions. After reverse transcription of 2 µg total RNA by using oligo(dT) primer, the resulting single-strand cDNA was amplified using Taq DNA polymerase (Promega Co.) and specific primers directed against human DLC1 (5-CAATGACGAAGAGAGTATGA-3’ and 5-TGGGAATAGATGTAGTAAAA-3’). PCR conditions were 95°C /2 min; 32 cycles at 94°C /30 sec, 55°C /30 sec, and 72°C /30 sec followed by 72°C /7 min 8


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for the final extension. Aliquots (10 µl) of the amplified cDNA were separated by 1.5% agarose gel electrophoresis,

and

visualized

by

ethidium

bromide

staining.

The

expression

level

of

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal control, primers used are: 5-AGTCGGATACACACATATTCATCA and 5-ATGGTGGGGTAGATCTTCTTCT-3’.

A real-time PCR assay was performed by using the same cDNA samples as templates. The reagent used was LightCycler® 480 Probe Master (Roche Ltd.). The reaction was performed and analyzed on StepOne™ real-time PCR system (Life Technologies Co.) using the same condition as RT-PCR, except that the amplification reaction was run for 50 cycles. The relative expression level (defined as fold △△Ct

change) of target gene DLC1 (2-

) was first normalized to the endogenous GAPDH reference (△Ct),

and then normalized to the expression level of DLC1 in NPs-DAC sample as 100%. Three independent experiments were performed to analyze the relative gene expression and each sample was tested in triplicate. Data were presented as mean ± SD. Statistical analysis was carried out with Student's t test. P < 0.05 was considered as statistically significant difference.

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Results Assembly and characterization of lipid-polymer nanoparticles Lipid–polymer hybrid NPs were prepared using a modified nanoprecipitation method [30-31] as described in Materials and Methods. All chemicals used for fabricating the NPs had been proved by Food and Drug Administration (FDA) for clinical use. As shown in Fig. 1A, the hybrid NPs are comprised of three components: a hydrophobic poly-(D,L-lactide-co-glycolide) (PLGA) core loaded with anti-cancer drugs, a hydrophilic poly(ethylene glycol) (PEG) shell, and an interphase between these two layers composed by soybean phosphatidylcholine (lecithin) monolayer. The PEG molecule is covalently attached to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE–PEG), which is interspersed throughout the lecithin monolayer. By changing the terminal group of DSPE–PEG, the NPs can be modified with different surface functional groups such as carboxyls, amines, and methoxyls.

In this study, we selected the traditional chemotherapeutic drug doxorubicin (DOX) to study the combinational effect with epigenetic drug 2’-deoxy-5-azacytidine (DAC). The NPs prepared for the following tests are NPs control, NPs-DOX, NPs-DAC, and NPs-DOX-DAC, indicating with no drug, DOX, DAC or both drugs encapsulated respectively. In a pre-test, the NPs encapsulation efficiencies for each drug or drug combination were measured as described in Materials and Methods (Fig. S1), and the drug concentrations used in preparing NPs were adjusted according to the desired final concentration. The NPs prepared had averaged sizes from 80.7 nm to 104.4 nm, which would allow the enrichment of NPs in tumor tissue through EPR effect 2. TEM image (Fig. 1B) also indicated that the NPs were dispersed as individual NPs with spherical shape and homogeneously size distribution. The averaged surface potentials of our NPs were from -28.65 mV to -34.10 mV (summarized in Table 1). Slightly negative charge on the NPs surface could protect NPs from clearance of the RES 2. Furthermore, to verify the stabilities of NPs, we quantitatively analyzed the amounts of free DOX in NPs-DOX-DAC suspension (Table S1). Our results showed that the NPs were very stable for at least 5 days, only less than 1% free DOX could be detected. And on day 20, still there were about 95% DOX kept encapsulated in NPs.

10


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DAC showed no synergistic promotion on DOX cytotoxity in free solution 5 µM DAC were used in this study as reported before

33

. A titration test was also done for DAC in

MB231 and HONE1 cell lines. As shown in Fig. S1, in free solution, DAC was able to repress the cell growth to 85% (MB231) or 90% (HONE1) at 5 µM. There is no significant increase in repression efficiency when the drug concentration was increased up to 250 µM. This indicates that the epigenetic drug, DAC, is a relative mild growth repressor compared to traditional chemotherapeutic drugs such as DOX. The inhibition of DOX on cell growth was then tested with or without 5 µM DAC (Fig. 2). DOX effectively inhibited cell growth at low concentration as 0.1 µg/ml. The growth inhibition increased with DOX concentration. After 72 hours incubation, less than 5% cells survived at the existence of 1 µg/ml DOX. However, the co-treatment with DAC didn’t significant affect the cell sensitivity to DOX at any concentration tested (Fig. 2C)

Co-delivery of DOX and DAC by NPs showed synergetic inhibition effect on tumor cell growth To test the suppression effects of our NPs on cancer cell growth, we first checked the uptake of NPs by cancer cells. In this cellular uptake assay, fluorescent dye Dio was incorporated into the interphase lipid monolayer of the NPs, thereby indicating the cellular uptake of NPs. After treated the MB231 cells with NPs-DOX-DAC+Dio for 2 hrs, the red fluorescence of DOX was observed inside the cancer cells and colocalized with the Dio fluorescence (Fig. 3A). This result suggested that the drug-encapsulated NPs were effectively captured by the cancer cells, therefore could be used for further growth inhibition assays. Moreover, most DOX was found inside the cytoplasm with 2 hrs treatment, indicating a slow releasing of DOX from NPs inside the cell. In another test, HONE1 and MB231 were treated with free DOX solution or NPs-DOX, and followed for 24 hrs (Fig. 3B and Fig. S3). The results indicated that the cells could continuously take in NPs for 24 hrs, while the cellular uptake of free DOX reached a plateau at around 12 hrs. The mean fluorescent intensity data also showed that the cells could take in NPs-DOX at higher efficiencies than free DOX. However, for both the DOX and NPs-DOX treatments, the quantitative HPLC analyses for total amount of DOX in the culture media showed that only about 20-30% DOX could be taken in by the cells in 24 hrs (data not shown). Nevertheless these data confirmed the NPs as an effective drug delivery tool. 11



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Growth inhibition effects of drug-encapsulated NPs were then tested in MB231 cell lines and compared with the effects of drug in free solution (Fig. 4). DAC in free solution or encapsulated with NPs (NPs-DAC) both showed mild effects on cell growth. Since nanoparticle packaging may limit the effective drug concentration exposed to the cancer cells, NPs-DOX, and NPs-DOX-DAC both showed less inhibition on cell growth than their free forms in solution. This property would allow higher drug dosage to be applied in treatment, without causing additional side effects. Interestingly, when DOX and DAC were co-encapsulated in the same NPs (NPs-DOX-DAC), it inhibited tumor growth more potently than either NPs-DOX or NPs-DAC. Since DAC in free solution didn’t enhance the cellular sensitivity to DOX treatment (Fig. 2C), the enhanced growth inhibition might be a combinational result of nanoparticle packaging and drug co-delivery effects.

Co-delivery of DOX and DAC by NPs could enhance the apoptosis of cancer cells In the growth inhibition assay, 1 µg/ml DOX was used to treat the cancer cells. At this DOX concentration the NPs-DOX-DAC might suppress cell growth to less than 30%. Here, a relative mild condition, 0.1µg/ml DOX was used to test the effects of NPs on cellular apoptosis in HONE1 cell line (Fig. 5A-E). After treated the cancer cells for 72 hours, apoptotic cells in culture incubated with NPs-DOX-DAC (30.93%) were much more than in cultures treated with PBS control (6.33%), NPs control (9.51%), NPs-DOX (14.43%), or NPs-DAC (15.18%) respectively.

We next checked the effect of DAC on the expression recovery of methylated TSG, DLC1, which had been shown to be silenced by DNA methylation in multiple human tumors 34-35. Expression of DLC1 was increased in HONE1 cells after DAC or NPs-DAC treatment, but not recovered in cells treated with NPs only (Fig. 5F). Interestingly, its expression in cells treated with NPs-DAC for continuous 72 hrs was stronger than in cells treated with DAC for 72 hrs without changing medium. Previous study had reported that the DAC was instable in the neutral aqueous buffers. The half life of DAC in pH7.0 potassium phosphate buffer under 37°C is less than 12 hrs

31

. Therefore, our data suggested that NPs

encapsulation might help stabilize DAC in the culture condition. Overall, these results further supported 12


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that NPs may serve as potential drug delivery tools in co-delivering multiple drugs at the same time; and that epigenetic therapy may increase the efficiency of cancer treatment when administrated together with traditional chemotherapeutic drugs.

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Discussion In this study, we aimed at the ability that NPs could pack and deliver multiple drugs at the same time. It was confirmed that the lipid-polymer nanoparticles were effectively applied on delivering epigenetic drug and traditional chemotherapeutic drug into cancer cells simultaneously. Our data clearly suggested that co-delivering both drugs exhibited much better repression effect on cancer cell growth than delivering single drug alone. The ratio of apoptotic cells in NPs-DOX-DAC treatment was also higher than that in single drug treatment.

Epigenetic drug is a novel type of anti-cancer drugs that can active the expression of a broad spectrum TSGs, while does much less damage to the normal cells compared to traditional chemotherapeutics. Among them, the DNA methylation inhibitors Aza and DAC had been approved for the treatment of myelodysplastic syndrome (MDS)

21

. Since many TSGs and genes involved in drug resistance were

silenced in tumor by epigenetic modification, the epigenetic drugs were valuable on enhancing cancer cell sensitivity to chemotherapeutic drugs. For an example, DAC was used to reverse drug resistance in cancer cell line A2780/cp70 lacking expression of DNA mismatch repair gene hMLH1 26. Restoration of hMLH1 through DNA demethylation sensitized the cancer cells to cisplatin, carboplatin, temozolomide and epirubicin. In other cases, DAC was used to restore the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and enhance the chemosensitivity of breast cancer cells to adriamycin

27

; DNMT inhibitor decitabine and an HDAC inhibitor were used together to induce the

expression of caspase-8 and sensitize drug resistant SCLC cells to TRAIL-induced apoptosis

25

.

Consistently in our experiments, when both DAC and DOX were delivered into the cell by NPs, enhanced growth inhibition and apoptosis were obvious than using single drug alone. In other reports, synergic growth inhibition or induced apoptosis were also observed. For an instance, a combination of DAC and paditaxel in solution forms shows synergic enhanced susceptibility of prostate carcinoma cells 28

. Notably, NPs by itself may promote cell sensitivity to the carried drugs: PLGA NPs is able to protect

its cargo from ABC mediated expelling

36

. Other mechanisms like NPs designed to respond to tumor

microenvironment may quickly release the drug inside the tumor cells and thus enhance the drug sensitivity 36. 14


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Another strategy in designing NPs, is to elongate the drug efficient period by limit the drug releasing speed 2. For traditional chemotherapeutic drugs, especially for those with very high cytotoxicities to normal cells, their clinical applications are greatly limited by the tolerance of the patients. Thus the dosages could be administrated to patients might not be enough to kill the tumor cells. Nanoparticle packaging is able to limit the effective drug concentration exposed to the cells, so that would allow more drugs to be taken by the patients and to overcome the tolerant threshold. In addition, a limited drug releasing process in combination with higher drug administration might also help to acquire a long term effect without causing unexpected side effects. In our tests, NPs-DOX and NPs-DOX-DAC did show much lower cytotoxicity than free DOX in solution, which was possible due to the lower effective concentration when using the lipid-polymer nanoparticles. Furthermore, in our drug co-delivery system, the epigenetic DAC showed obvious synergetic promotion effect on the chemotherapeutic drug DOX. This suggested that: in the existence of DAC, once the DOX was released and exposed to the cancer cells, its ability to inhibit cancer cell growth was higher than the free DOX at the similar effective concentration. We should also noticed that a slow releasing of epigenetic drugs might be an advantage, since epigenetic alternation induced by these drugs usually takes generations to appear. Thus longer efficient period may enhance the synergic enhancement of epigenetic therapy on chemotherapy. Overall, our nanoparticle co-delivery system may provide two advantages on enhancing the effectiveness of chemotherapy: (1), nanopackaging effect would allow more chemotherapeutic drugs to be administrated; (2), the existence of epigenetic drug would enhance the sensitivity of tumor cells to chemotherapeutic drugs. With this potential tool in hand, we should be able to perform further in vivo works on tumor suppression in experimental animals. In one of the studies in our research group, the folate conjugated lipid-polymer NPs were injected into nude mice via the tail vein. These NPs could effectively accumulate in the xenotransplanted tumors 30. Similar work would be performed for our NPs-DOX-DAC in the near future.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81071634, 81101492, 81071249, 81171446), Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies (GIRTF-LCHT), the Key Project of Science and Technology of Guangdong (2009A030301010), the Shenzhen Science and Technology Program (Grant No. JC20100570328A, JC201005260247A, CXB201005250029A), and the “Hundred Talents Program” of Chinese Academy of Sciences.

16


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Table 1: Size and surface Z-potential of functionalized lipid-polymer hybrid NPs. Nanoparticles (NPs)

Size

Zeta potential

NPs

93.6±24.5 nm

-34.10 mV

NPs-DAC

91.3±25nm

-28.65 mV

NPs-DOX

104.4±27.6nm

-32.78mV

NPs-DOX-DAC

80.7±20.1nm

-34.07mV

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Figures & figure legends: A

B

PLGA DOX DAC Soybean lecithin DSPE-PEG-COOH

Figure 1 (A) Schematic illustration of lipid-polymer hybrid nanoparticle (NP) bearing encapsulated chemotherapeutic drug DOX, and epigenetic drug DAC. The NP was comprised of a poly (D,L-lactide-co-glycolide) (PLGA) core, a polyethylene glycol (DSPE-PEG-COOH) shell and a lipid monolayer (Soybean lecithin) at the interface of the core and the shell. (B) A negative stain TEM image of the NPs-DOX-DAC (x40000).

18


Page 19 of 27

B

2.0

24hr 48hr 72hr 96hr

OD490

1.6 1.2 0.8

24hr 48hr 72hr 96hr

1.2

0.8

0.4

0.4 0.0

0.0

0

0.05 0.1 0.25 0.5

1

1.5

2

DOX concentration (µg/ml)

C

1.6

OD490

A

120%

Cell Survival (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

0.05 0.1 0.25 0.5

1

1.5

2


DOX DOX+DAC

80%

40%

0% 0

0.05 0.1 0.25 0.5

1

1.5

2


Figure 2 Human breast carcinoma cell line MB231 were treated with various doses DOX in free solution for 96h. Cell proliferation was determined by MTS assays every 24 hrs. DOX dose titration 0, 0.05，0.1，0.25，0.5，1，1.5，2 µg/mL were used in combination without (A) or with (B) 5 µM DAC. C. After treated with the drugs for 72 hrs, cell survival rates were normalized to OD490 of culture treated with no drug as 100%. DAC showed no significant effect on the cytotoxicity of DOX at all the concentrations tested.

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A

Dio

Hoechs

DOX

Merge

NPs+Dio

NPs-DOX

NPs-DOX-DAC+Dio

B

80000

Mean FL2

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HONE1+DOX HONE1+NPs-DOX MB231+DOX MB231+NPs-DOX

60000

40000

20000

0 0

5

10

15

20

25

Time (hr) Figure 3 Uptake assays for nanoparticles. (A) Confocal images of MB231 cells incubated with nanoparticles for 2 hrs. The nuclei were stained with Hoechs 33258 for 10 min (blue channel). Green and red fluorescence was from Dio lipid dye and DOX respectively. Upper panel showed cells treated with NPs control, Dio fluorescent dye was incorporated in NPs (NPs+Dio) to show the uptake of NPs. Middle panel showed cells treated with NPs-DOX, red fluorescence of DOX was used to monitor the uptake of NPs-DOX. Lower panel showed cells treated with NPs-DOX-DAC+Dio, DOX and Dio were colocalized in the cytosol of these cells. DOX concentration used in this test were 1 µg/ml. Scale bars 20


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equal 25 µm. (B) Quantitative uptake analyses for HONE1 and MB231 cells treated with DOX or NPs-DOX (DOX final concentration of 0.2 µg/ml). Mean fluorescent intensities of DOX (FL2) in the cells were measured by flow cytometry and used to indicate the accumulation of DOX inside the cells.

21



PBS NPs DOX DAC DOX+DAC NPs-DOX NPs-DAC NPs-DOX-DAC

1.2 1

OD 490 nm

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0.8 0.6 0.4 0.2 0 24

48

72

96

120

Time (hr)

Figure 4 Effects of DOX (1 µg/ml), DAC (5 µM) and various nanoparticles on the growth of human breast carcinoma MB231 cell line. Cell growth was determined by MTS (OD 490 nm) at every 24 hrs for 5 days. PBS (filled circle) was used as treatment control. NPs without drug (open circle), NPs-DAC (open diamond), and DAC in free solution (filled diamond) showed mild effects on cell growth. DOX in free solution (filled triangle) or in combination with 5 µM DAC (open square) showed the highest repression on cell growth. Nanoparticles encapsulated DOX (NPs-DOX, open triangle) significantly decreased the cytotoxicity of free DOX. This relative mild cell growth repression effect of NPs-DOX could be synergistically promoted in the co-existence of DAC (NPs-DOX-DAC, filled square).

22


B

PBS

9.51%

C

NPs

NPs-DOX

E

F NPs

D

14.43%

15.18%

30.93%

NPs-DAC

6.33%

DAC-2

A

DLC1 GAPDH 1.2 0.8 0.4

D AC Ps 2 -D AC

1

N

NPs-DOX-DAC

AC

NPs-DAC

N

Ps

0

D

Normalized DLC1 expression

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DAC-1

Page 23 of 27

Figure 5 Effects of the treatments with nanoparticles containing DOX (0.1 µg/ml), DAC (5 µM) or in combination on apoptotic cell death. HONE1 cells were incubated with PBS control (A), NPs without drug (B), NPs-DOX (C), NPs-DAC (D), or NPs-DOX-DAC (E) for 72 hrs. Cells were then collected and stained with Annexin V FITC Apoptosis Detection Kit. The red fluorescence (FL2, Y axis) of propidium iodide stained the DNA of the dead or late apoptotic cells, and the green fluorescence (FL1, X axis) of FITC conjugated annexin V stained the phosphatidylserine on the outer membrane of early apoptotic cells (lower-right region of each small panel) and late apoptotic cells (upper-right region of each small panel). Total apoptotic cells percentages were calculated including both early and late apoptotic cells and were shown on each panel. (F) HONE1 cells were treated with NPs control, DAC or NPs-DAC as described in Materials and Methods, whereas DAC-1 means the culture was changed with fresh medium containing 5 µM DAC every 24 hrs, DAC-2 means culture treated with 5 µM DAC for continuous 72

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hrs without changing medium. The RT-PCR and Real-time PCR results showed the expression recovery of tumor suppressor gene DLC1 in cells treated with DAC or NPs-DAC. GAPDH was used as internal control. Compared to the DLC1 expression level of NPs-DAC sample, the P values of NPs, DAC1, DAC2 samples were 1.12x10-7, 4.13x10-3, and 3.29x10-5 respectively.

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LipidâPolymer Nanoparticles Encapsulating Doxorubicin and 2

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