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The Promising Nanocarrier for Doxorubicin and siRNA Codelivery by PDMAEMA Based Amphiphilic Nanomicelles Qiang Cheng, Lili Du, Lingwei Meng, Shangcong Han, Tuo Wei, Xiaoxia Wang, Yidi Wu, Xinyun Song, Junhui Zhou, Shuquan Zheng, Yuanyu Huang, Xing-Jie Liang, Huiqing Cao, Anjie Dong, and Zicai Liang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11789 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 3, 2016

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ACS Applied Materials & Interfaces

The Promising Nanocarrier for Doxorubicin and siRNA Co-delivery by PDMAEMA Based Amphiphilic Nanomicelles

Qiang Cheng,*,†,┴ Lili Du,†,┴ Lingwei Meng,† Shangcong Han,‡ Tuo Wei,# Xiaoxia Wang,† Yidi Wu,† Xinyun Song,† Junhui Zhou,‡ Shuquan Zheng,† Yuanyu Huang,† Xing-jie Liang,# Huiqing Cao,† Anjie Dong,‡,§ and Zicai Liang†,§ †

Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University,

Beijing 100871, China ‡

Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering

of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China §

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China #

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

Nanoscience and Technology of China, Beijing 100190, China

ABSTRACT Synergistic effects of anticancer drug and siRNA has displayed superior advantages for cancer therapy. Herein, we deeply analyzed the feasibility that whether doxorubicin (DOX) and siRNA could be co-delivered by mPEG-PCL-graft-PDMAEMA (PECD) micelles, which mediated excellent DNA/siRNA delivery in vitro and in vivo reported in our previous work. DOX-loaded NPs (PECD-D) were developed by nanoprecipitation technology and exhibited 1

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high drug loading content (DLC, 9.5%). In vitro cytotoxicity study in MDA-MB-231 cells, PECD-D treated groups had lower IC50 compared to free DOX groups (F-DOX) at different transfection time (24h, 48h and 72h), which maybe attribute to its high cellular uptake and endosomal escape properties. The speculation was confirmed with the results of drug release profile in acidic media, flow cytometry analysis and confocal images. Futhermore, Cy5 labelled siRNA was introduced in PECD-D micelles (PECD-D/siRNA) to track the behavior of dual-loaded nanodrug in vitro and in vivo. Flow cytometry analysis presented that DOX and siRNA were successfully co-delivered into cells, the positive cells ratio were 94.6% and 99.5%, respectively. Confocal images showed that not only DOX and siRNA existed in cytoplasm, but DOX traversed endosome/lysosome and entered into cell nucleus. For in vivo tumor-targeting evaluation in BALB/c nude mice, both DOX and Cy5-siRNA cuould be detected in tumor site after intravenous injection with PECD-D/siRNA formulation. Therefore, we believed that PECD micelles had potential ability as DOX and siRNA co-delivery carrier for cancer therapy. KEYWORDS: co-delivery, PDMAEMA, nanodrug, siRNA, doxorubicin.

INTRODUCTION Despite considerable progress on cancer therapy, tumors remain one of the major causes

of mortality and morbidity in the world, about more than ten million new cases every year1. As common treatment means, chemotherapy is choiced in many cancers. However, its success has been limited by several drawbacks. Take doxorubicin (DOX) as the example, since broad-spectrum antineoplastic drug, DOX is used to treat many kinds of tumors in clinical, 2

3

4

such as lung cancer , hepatocellular carcinoma and breast cancer . But the side-effects are 2


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also obvious, for example cardiotoxicity5, typhlitis, nausea6 and so on. Doxorubicin also presents nonspecific distribution and short blood half-life

7-8

after intravenous administration,

making them easily be exposed on normal organs and fast excreted by kidney, leading to systemic toxicity and bad effect when curing cancers. What’s worse, multidrug resistance (MDR) further hinders improvement of anticancer effect9-10 during treatments period. To overcome the barriers, many kinds of DOX-loaded nanoparticles (NPs), such as liposomes11, 12

polymer-based micelles , lipid-based micelles

4, 13

, conjugates carriers

14

and inorganic

nanoparticles15, have been fabricated and tested by researchers. These NPs could accumulate in tumor tissues via enhanced permeation and retention (EPR) effect, a well known phenomenon that NPs bearing suitable size (smaller than 200nm) are inclined to leak out from tumor vessels and arrive at tumor tissues due to tumor defective architecture of blood 16-18

vessels

. Poly(ethylene glycol) (PEG) decorated NPs significantly extend DOX blood

half-life and improve treatment effect19. Additionally, tumor active targeting of NPs through 20

21

receptor-mediated endocytosis, such as folate , hyaluronic acid (HA) , prostate-specific membrane antigen (PSMA) aptamer22 and iRGD23, has shown potential application value for tumor-targeting delivery. Thus, tumor-specific delivery has been realized with smart DOX-loaded nanoparticles by passive (EPR effect) or (and) active (receptor-mediated) targeting. At the same time, bad pharmacokinetics and systemic toxicity limitations of DOX has been improved. Furthermore, DOX-loaded nanomicelles can combat multidrug resistance (MDR) by avoiding the pump resistance mechanism4 that DOX is pumped out from cells by membrane transporters24. To date, gene therapy is regarded as a promising strategy for cancer therapy, especially 3



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for small interfering RNA (siRNA)25, which can be incorporated RNA-induced silencing 26

complex (RISC) and degrade target message RNA (mRNA), called RNA interference (RNAi) . Although siRNA could be used to specifically and effectively silence disease genes, the application has been limited. SiRNA is instability and almost could not be endocytosed by cells due to large molecular weight (~13 KDa) and strong negative charge27. One of the best methods to solve these disadvantages is using delivery vehicles. In comparison, non-viral 25

carriers have great advantages than viral carriers in safety, large-scale production and cost . Among the various non-viral vectors, cationic polymers are attractive and effective for siRNA delivery in vitro and in vivo, such as polyethyleneimine (PEI) methacrylate

(GEMA)31,

poly

(L-lysine)

(PLL)32,

28-29

30

, chitosan , 2-guanidinoethyl

polyamidoamine

(PAMAM)33

and

2-(dimethylamino) ethyl methacrylate (DMAEMA)34. Among them, DMAEMA has been considered as excellent DNA/siRNA carriers since low cytotoxicity and buffering capatity (known

as

proton

sponge

effect)

for

endosomal

escape35.

Besides,

poly

(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) exhibits other advantages, such as copolymer could be easily synthesized by atom transfer radical polymerization (ATRP) or reversible addition fragmentation transfer (RAFT), molecular weights are controlled and 36

different architectures (such as block and graft) could be fabricated . In our previous work, a series of PDMAEMA-based amphiphilic cationic polymers have been developed and evaluated on DNA/siRNA delivery. All of them are consist of PEG, hydrophobic core and PDMAEMA.

Based

on

the

first

generation

glycol)-block-(polycaprolactone-graft-poly(2-(dimethylamino)ethyl

vector,

poly(ethylene

methacrylate))

(PECD),

many kinds of modifications were used to improve delivery efficiency or to study delivery 4


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mechanism, which contains structure change of PDMAEMA37, folate-decorated ternary 38

34

39

complexes , different hydrophobic core components , glutathione (GSH) sensitive

and

mesoporous silic functionalized copolymers40. However, whether the PECD micelles could be used to deliver anticancer drug is not discussed yet. Synergistic effects of anticancer drug and siRNA for cancer treatment have deeply catched researchers’ attention in resent years41. One side, dual-loaded NPs improve chemotherapeutic effect by affecting drug resistance channels, ⅰ ) pump resistance mechanism: keeping away from membrane transporters by NPs and silencing related gene (e.g. P-glycoprotein, P-gp) by siRNA; ⅱ) nonpump resistance mechanism: shutting down the antiapoptotic gene (e.g. BCL-2) by siRNA42. The other side, dual-loaded nanodrug achieve duplicate effect on cell antiproliferation for cancer therapy by inhibition mitosis-related genes 43

(e.g. polo-like kinase 1, PLK1) with siRNA . In this work, we aimed to explore the new application value of PECD micelles. To answer the question that whether PECD could be used to co-deliver DOX and siRNA, feasibility analysis was performed. As illustrated in Scheme 1, dual-loaded nanodrug was prepared by combining nanoprecipitation technology and electrostatic interaction (Scheme. 1A). Schematic presented PECD mediated siRNA and DOX co-delivery in cells (Scheme. 1B). In this research, we chose MDA-MB-231 cells as the model.

5



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Scheme 1. Schematic illustration of dual-loaded nanodrug and their dynamic process in cells. (A) Amphiphilic cationic polymer (PECD) was synthesized by combining ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP), DOX-loaded micelles (PECD-D) were prepared by self-assemble using nanoprecipitation technology, dual-loaded nanoparticles (PECD-D/siRNA) were developed via electrostatic interaction. (B) Schematic presentation of PECD mediated siRNA and DOX co-delivery in cells. NPs were internalized by endocytosis (ⅰ), undergoing endosomal acidification and proton sponge effect of DMAEMA (ⅱ), finishing endosomal escape and drug release (ⅲ), DOX acted on DNA by nucleic pathway (ⅳ) and siRNA silenced target mRNA by RNAi pathway (ⅴ).

EXPERIMENTAL SECTION Materials. Methoxy poly(ethylene glycol) (mPEG, Mn=2kDa), 2-(dimethylamino) ethyl

methacrylate (DMAEMA), ε-caprolactone (CL), copper(I) bromide (CuBr), Stannous octanoate (Sn(Oct)2), and N,N,N',N'',N''-pentamethyl diethylenetriamine (PMDETA) were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (DOXHCl) was purchased from Zhejiang Hisun Pharmaceutical Co. Ltd. 6


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Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, trypsin, penicilin-streptomycin and fetal bovine serum (FBS) were purchased from Invitrogen Corporation (Carlsbad, CA). Agarose was purchased from GEN TECH (Hong Kong, China). Ethidium bromide (EB), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium

bromide

(MTT),

2,2,2-trifluoroethanol

(TFEA), Sodium tetraborate (Na2B4O710H2O) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Cy5-labelled siRNA (Cy5-siRNA) and scrambled siRNA (siNC) were supplied by Suzhou Ribo Life Science Co., Ltd (Suzhou, Jiangsu Province, China), both of

them

shared

same

sequences

5’-UUCUCCGAACGUGUCACGUdTdT-3’;

as

follows:

sense

antisense

strand, strand,

5’-ACGUGACACGUUCGGAGAAdTdT-3’. Cy5 fluorophore was labelled at 5’ of the sense strand. All other reagents were analytical pure and used without further purification.

Synthesis of PECD Polymer. Ƴ-(2-bromo-2-methylpropionate)-ε-caprolactone (BMPCL) was synthesized as reported previously34, 44. mPEG-PCL-g-PDMAEMA (PECD) was synthesised by combining ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP) as described before34. Briefly, BMPCL and ε-caprolactone were used to synthesis the macroinitiator, named mPEG-P(CL-co-BMPCL) (PECB), by ring-opening polymerization with mPEG as the initiator and Sn(Oct)2 as the catalyst. This reaction was stirred at 130℃ for 10h in a nitrogen-purged dry schlenk flask. Then the ATRP polymerization in presence of PECB and DMAEMA was performed at 60℃ for 24h with butanone as solvent then gained PECD. The products were passed through a basic aluminum oxide column to remove the catalyst and dialyzed against distilled water for 24h. Then the purified copolymer 1

was freeze-dried to prepare to use. Finally, PECD was characterized by H NMR (Varian 7



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Unity-Plus INOVA 500).

Preparation of Nanoparticles. Deprotonated DOX was achieved by the following method. Briefly, DOXHCl (200mg) was dissolved in double distilled water, then sodium tetraborate (760mg) was added into water and stirred for 4h. Subsequently, suspension was centrifuged for 10min at speed of 5000rpm and collected precipitation. After washed three times with water, deprotonated DOX was freeze-dried and stored in -20℃. The empty PECD (PECD-E) was prepared by nanoprecipitation technology. In detailed, PECD (30 mg) was dissolved in TFEA (1.5 ml), added dropwise into deionized water (8 ml) under vigorous stirring. This solution was dialyzed in deionized water using dialysis tubing (Spectra/Por, Mw cut-off:10kDa) by frequently exchange water to completely remove TFEA, final volume was adjusted to 10 ml. Doxorubicin (DOX)-loaded PECD (PECD-D) was prepared as the same as PECD-E did except for adding deprotonated DOX (3.6 mg) in TFEA together with PECD and centrifuging at speed of 4000rpm for 10min to remove unencapsulated DOX after finishing dialysis. These NPs were stored at 4℃. For PECD-E/siRNA and PECD-D/siRNA preparation, the PECD-E or PECD-D suspension was added into equal volume siRNA solution using pipetting and incubated for 20min at room temperature. Before preparation, the amount of NPs and siRNA were calculated according to the siRNA transfection concentration and N/P ratio.

Physicochemical Properties. The size distribution and zeta-potential of PECD-E and PECD-D were measured by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern, UK) at a wavelength of 633 nm with a constant angle of 173 at room temperature. These NPs suspension were prepared for 1ml volume contained 3mg/ml copolymers. The sizes and 8


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zeta-potential of PECD-E/siRNA and PECD-D/siRNA were also detected by DLS but contained 2µg siRNA at the N/P ratio of 6.7:1.

Determination of Encapsulated DOX. To measure encapsulated DOX in PECD NPs. Freeze-dried PECD-D was weighted and dissolved in DMSO, then fluorescence intensity was determinated by Multi-Mode Microplate Reader (Synergy HT, BioTek, USA) with 485nm excitation and 592nm emission. A calibration curve was constructed using different free DOX concentrations (0-20 µg/ml) in DMSO. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following equations: DLC%=

weight of loaded DOX ×100 weight of DOX―loaded NPs

DLE%=

weight of loaded DOX ×100 weight of DOX in feed

DOX Release. DOX release from PECD-D was determinated by dialysising method. Briefly, 1 ml PECD-D solution (1 mg/ml) was sealed in dialysis bag (Mw cut-off:10kDa) and incubated in 30ml PBS (pH=5.0 and pH=7.4) at 37℃ under shaking at the speed of 200rpm. After 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, 60h and 72h, 1ml released PBS was withdrawn for testing and replaced with equal volume of fresh PBS. The DOX fluoresence intensity was detected by Multi-Mode Microplate Reader and the conecntration was calculated according to calibration curve. The accumulative released was calculated by the following equation: E%=

Va ∑n-1 1 Cb +Vi Cn ×100 M

Where E(%) represnts accumulative released percentage, MDOX represents the total amount of DOX in the NPs, Vi is the whole volume of the release media (Vi=30ml), Va is the volume of the replaced media (Va=1ml), Cn represents the concentration of DOX of the nth sample and Cb st

th

represents the concentrations of DOX from 1 to (n-1) samples. Three groups of replicate 9



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measurements were carried out for each time point.

Cell Viability. MDA-MB-231 cells were seeded at 5 ×103 cells per well in a 96-well plate the day before transfection. Cells were treated with F-DOX (free doxorubicin) and DOX-loaded PECD (PECD-D) at DOX concentrations ranging from 0.1 to 50µg/ml under Opti-MEM medium for 6h. To exclude the influence of polymer for cytotoxicity, cells were also treated by PECD-E alone for the same time at different concentrations. Then Opti-MEM medium was removed and replaced with fresh DMEM to further incubate 24h, 48h and 72h, respectively. After that, the medium was replaced with 100µl 0.5mg/ml MTT for 4h and the MTT solution was replaced with 50µl DMSO for another 10min at 37℃. The absorbance was measured at 540nm (OD540) with a reference wavelength of 650nm (OD650) using Multi-Mode Microplate Reader. Untreated cells were used as control (mock). All experiments were carried out with three replicates. The cell viability (%) was calculated by following equation: Cell viability%=

OD − OD ×100 OD − OD

Gel Retardation assay. NPs/siRNA complexes were prepared as reported previously from our laboratory. Briefly, PECD-E or PECD-D solution was mixed with 0.3µg scrambled siRNA (siNC) at various charge ratio (N/P ratio) for 20min at room tempreture, then adjusted to final 16µl volume. Four µl of 6× loading buffer (Takara Biotechnology, Dalian, Liaoning Province, China) was added into the NPs/siNC polyplexes suspension, then 20µl of the mixture was loaded onto 2% agarose gel containing 5µg/ml ethidium bromide (EB). Electrophoresis was carried out at 120V voltage for 20min in 1×TAE running buffer. Finally, the results were analyzed with image master VDS thermal imaging system (Bio-Rad, Hercules, CA) at UV light wavelength 254 nm. The charge raito (N/P ratio) means the molar ratio of 10


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amino group (N) of DMAEMA to phosphate group (P) of siRNA phosphate backbone.

Fluorescence-Activated Cell Sorting. To assess endocytosis ability of PECD-E/siRNA and PECD-D/siRNA complexes by cells. Fluorescence labeled siRNA was 5

used. MDA-MB-231 cells were seeded at 2 ×10 cells per well in 6-well plates. After 24h, the DMEM medium was replaced by Opti-MEM and cells were incubated with PECD-E/Cy5-siRNA and PECD-D/Cy5-siRNA polyplexes for 2h at the final DOX concentration of 1µg/ml and Cy5-siRNA concentration of 100nM, respectively. Cells were digested by tripsin (0.25%), washed three times by 1ml cold PBS, resuspended in 400µl PBS and analyzed by FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

Free DOX (F-DOX) and free

Cy5-siRNA (F-siRNA) treated cells were the control groups.

Subcellular Localization Studies. For subcellular localization studies of NPs/siRNA formulations, fluorescence labeled siRNA was also used. Briefly, MDA-MB-231 cells (2 ×105 cells per dish) were seeded into 35mm dishes the day before transfection, then treated with PECD-E/Cy5-siRNA and PECD-D/Cy5-siRNA polyplexes for 2h in Opti-MEM medium at the final DOX concentration of 2µg/ml (or 1µg/ml) and Cy5-siRNA concentration of 100nM, respectively. Subsequently, cells were imaged using Zeiss confocal microscope (LSM700, Carl Zeiss, Germany). F-siRNA, F-DOX and PECD-D treated cells were used to as the corresponding controls. Hoechst 33342 was used to stain nucleus to assist subcellular 34

localization analysis.

Pharmacokinetics And Biodistribution. All the animals used in this research were maintained in Peking University Laboratory Animal Center (an AAALAC-accredited experimental animal facility). All procedures involving experimental animals were performed in 11



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accordance with protocols approved by the Institutional Animal Care and Use Committee of Peking University. For pharmacokinetics studies, male C57BL/6j mice, weighting 18-22g, were purchased and randomly divided into three groups. Animals were administrated with F-DOX and PECD-D/siNC at the given dose of DOX (5mg/kg) and siRNA (6.65mg/kg, N/P=6.7) via intravenously (i.v.) injection, respectively. PBS treated mice were divided into the control group and every group contained three mice. The blood samples (50µl) from orbital venous were collected in Ethylene Diamine Tetraacetic Acid (EDTA) treated tubes at different time points (1, 5, 15, 30, 60 and 120 minutes) after injection, plasma was separated by centrifuging at the speed of 4000rpm for 20 min. For DOX measurement, DOX was firstly extracted from plasma as described previously45. Briefly, 10µl of plasma sample and 490µl of acidified 90% isopropanol (0.1M HCl) were mixed overnight at 4 ℃ packed by aluminum foil. The isoproponal extract was collected by centrifuging at 12000rpm for 10min at 4℃. Then DOX concentration was detected by Multi-Mode Microplate Reader and calculated via standard curve as described above. Twenty-four hours after injection, the mice were sacrificed by cervical dislocation, and main organs were used to further extract and quantify of DOX. Briefly, the organs were weighted, suspend in acidified 90% isopropanol (0.1M HCl) (100mg/ml) and homogenized, incubated overnight at 4℃. DOX suspension was received by centrifuging at the speed of 12000rpm for 10min at 4 ℃ and collected the supernatent. DOX concentration was determinated according to the protocol described above.

Tumor Accumulation Studies. To evaluate tumor-targeting ability, MDA-MB-231 12


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(5×106 cells) were suspended in 100µl PBS and injected subcutaneously in right axillary fossa 3

of female BALB/c nude mice. When the tumor grew to about 400 mm , the mice were divided into three groups according to the volume of tumors to make sure the tumors sizes were similar among groups. Mice were injected intravenously with PBS, F-siRNA (Cy5 labelled), and PECD-D/siRNA (Cy5 labelled) at the given dose of DOX (5mg/kg) and siRNA (6.65mg/kg), respectively. The Cy5 fluorescence signals of the whole body was observed using Kodak in vivo imaging system (Kodak In-Vivo Imaging System FX Pro, Carestream Health, USA) at different time points. At the endpoint, mice were sacrificed by cervical dislocation, and the major tissues including tumors were isolated and further examined using the Kodak in vivo maging system. In addition, the DOX in tumors was performed to extract and analyze as did as described last section.

Statistical analysis. Statistical analysis was performed by GraphPad Prism 5 software. Data were expressed by Mean ± SEM (standard error of mean). For statistical comparisons, two-tailed student’s t-test was employed, P

The Promising Nanocarrier for Doxorubicin and ... - ACS Publications

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