Biomaterials 35 (2014) 9495e9507

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Delivery of siRNA by MRI-visible nanovehicles to overcome drug resistance in MCF-7/ADR human breast cancer cells Gan Lin a, 1, Wencheng Zhu a, 1, Li Yang a, Jun Wu a, Bingbing Lin a, Ye Xu a, Zhuzhong Cheng a, Chunchao Xia b, Qiyong Gong b, Bin Song b, Hua Ai a, b, * a b

National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, China Department of Radiology, West China Hospital, Sichuan University, Chengdu 610041, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2014 Accepted 23 July 2014 Available online 23 August 2014

Multidrug resistance (MDR) is one of the major barriers in cancer chemotherapy. P-glycoprotein (P-gp), a cell membrane protein in MDR, also a member of ATP-Binding cassette (ABC) transporter, can increase the efflux of various hydrophobic anticancer drugs. In this study, polycation/iron oxide nanocomposites, were chosen as small interfering RNA (siRNA) carriers to overcome MDR through silencing of the target messenger RNA and subsequently reducing the expression of P-gp. Amphiphilic low molecular weight polyethylenimine was designed with different alkylation groups and alkylation degree to form various nanocarriers with clustered iron oxide nanoparticles inside and carrying siRNA through electrostatic interaction. A few optimized formulations can form stable nanocomplexes with siRNA and protect them from degradation during delivery, and lead to effective silencing effect that comparable to a commercial golden standard transfection agent, Lipofectamine 2000. Human breast cancer MCF-7/ADR cells can be vulnerable to doxorubicin treatment after the strong downregulation of P-gp through siRNA tranfection. Once transfected with these nanocomplexes, the cells displayed significant contrast enhancement against non-transfected cells under a 3T clinical MRI scanner. These nanocomposites also demonstrated their downregulation efficacy of P-gp in a MCF-7/ADR orthotopic tumor model in mice. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Multidrug resistance P-glycoprotein Superparamagnetic iron oxide Small interfering RNAs Magnetic resonance imaging

1. Introduction Chemotherapy remains as the main conventional option to treat cancer. However, multidrug resistance (MDR) in cancer cells makes the therapeutic efficacy less effective [1,2]. One of the most studied mechanisms about MDR is the increased efflux of various hydrophobic cytotoxic drugs, which is mediated by a family of energydependent transporters known as ATP-binding cassette (ABC) transporters [2]. Among them, P-glycoprotein (P-gp; MDR1), stands out as it confers strong resistance to many well-known chemotherapy compounds [3,4]. It is considered that drug resistance could be avoided by inhibiting P-gp function through chemical inhibitors. Although different generations of P-gp inhibitor look promising in cell models, they have little success in reversing MDR in clinical trials

* Corresponding author. National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. Tel./fax: þ86 28085413991. E-mail addresses: [email protected], [email protected] (H. Ai). 1 These authors contributed equally in this work. http://dx.doi.org/10.1016/j.biomaterials.2014.07.049 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

because of some unwanted side effects and unpredictable pharmacokinetic interactions with anticancer drugs [2,4]. Apart from chemical P-gp inhibitor, small interfering RNAs (siRNAs) could become a more powerful method to reverse MDR in cancer cells [5e7]. siRNAs are short molecules of 21e25 nucleotides that can trigger silencing of homologous gene expression by unwinding the siRNA duplex through an RNA-induced silencing complex (RISC), ultimately inducing recognition and degradation of a target messenger RNA (mRNA). So far, siRNAs have been used to knockdown the expression of MDR-related proteins by silencing Pgp, MRP1, and Bcl2 gene in MDR cancer cells [8e10]. Compared with small molecule P-gp inhibitors, the advantages of siRNAs are its high specificity and its reduced toxicity on non-specific tissues. However, its application faces many obstacles, such as ribonuclease (RNase) degradation, elimination, poor permeability, and endosomal trapping. Molecular imaging noninvasively visualizes and quantifies biological processes at molecular and cellular levels in a real-time way. Furthermore, it enables repetitive imaging in the same subject, thereby provides more detailed information by harnessing the statistical power of longitudinal studies [11,12]. There are a few

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types of nanocarriers which have been developed and loaded with drugs and imaging agents, to predict and monitor therapeutic responses [12,13]. Magnetic resonance imaging (MRI) has several advantages over other imaging modalities, including high spatial resolution, deep tissue penetration, excellent soft tissue contrast, and without using of radioisotopes or X-rays [14e17]. In our previous work, low molecular weight amphiphilic alkylated PEI (Alkyl-PEI) encapsulated superparamagnetic iron oxide (SPIO) nanoparticles were used for efficient siRNA and plasmid DNA delivery [18,19]. These polycation covered SPIO nanoparticles provide good biocompatibility and effective biomolecule protection. At the meantime, these nanocomposites demonstrated good contrast enhancement under MRI. However, how structural details of Alkyl-PEI will have impacts on their transfection functions need systematic investigation. In this study, we designed and synthesized a series of Alkyl-PEI, and investigated their critical micelle concentration (CMC), binding ability and gene silencing efficiency. A few formulations were chosen to form the Alkyl-PEI/SPIO nanovehicles. And as shown in Scheme 1, nanocomplexes of negatively charged P-gp-siRNA and positively charged Alkyl-PEI/SPIO were formed through electrostatic interactions. In our hypothesis, these MRI-visible nanovehicles may deliver P-gp-siRNA to MCF-7/ADR cells, down-regulate the expression of P-gp significantly and show obvious MRI contrast effects under clinical MRI scanner. 2. Materials and method 2.1. Materials 1-Iodohexane (98%), 1-Iodododecane (98%), 1-Iodooctadecane (95%), 1,2Hexadedecanediol(90%), Iron(III) acetylacetonate (99.9%), oleylamine (70%), oleic acid (90%), 1-Octadecen(90%), Fluorescein isothiocyanate (FITC) and Hoechst 33258 (98%) and human recombinant insulin (98%) were purchased from SigmaeAldrich (USA). Polyethylenimine (PEI, branched, Mw 1.8kD) were purchased from Alfa Aesar (USA). Dulbecco's modified eagle's medium (DMEM), RPMI-1640, penicillin/streptomycin, L-glutamine and fetal bovine serum (FBS) were purchased from Hyclone (USA). siRNA for P-gp knockdown and nontargeted-siRNA were purchased from RiboBio (Guangzhou, China). b-actin and P-gp antibodies were from Santa Cruz Biotechnology, Inc (USA). Cell Counting Kit-8 assay and Cy5-Labeled Goat Anti-Mouse IgG were obtained from Beyotine Institute of Biotechnology (Jiangsu, China). Lipofectamine™ 2000 Reagent was purchased from Invitrogen (USA). Doxorubicin hydrochloride (Dox, 98%) was from Taizhou Bolon Pharmchem (Shanghai, China). RAW264.7 and MCF-7/ADR (multidrug resistant human breast cancer cell line) cells were obtained from West China School of Pharmacy Sichuan University (Chengdu, China). RAW264.7 cells were cultured in RMPI-1640 with 10% FBS and 1% penicillin/streptomycin. MCF-7/ADR cells were cultured in DMEM with 10% FBS and 1% penicillin/ streptomycin. All cells were incubated in a humidified atmosphere of 5% CO2 at 37  C with the medium changed every other day. And to maintain the MDR property of MCF-7/ADR cells, it's necessary to add 1 mg/mL doxorubicin in the medium before experiments. BALB/c nude female mice (6e8 weeks age, average body weight 18e20 g) were purchased from Sichuan University Laboratory Animal Center (Chengdu, China).

2.2. Alkylation of PEI2k Alkyl-PEIs were synthesized following a published protocol [20]. Briefly, hyperbranched PEI were reacted with 1-Iodohexane (1-Iodododecane or 1Iodooctadecane) in refluxing CHCl3 in the presence of potassium carbonate. The crude products were dissolved in alcohol, and dialyzed against it, then thrice against water. Pure products were obtained after lyophilization, and the degree of alkylation was determined by elemental analysis.

2.4. Differential scanning calorimetry (DSC) DSC measurements were carried out using TA DSC Q2000 (USA) under nitrogen atmosphere in the temperature ranging from 80 to 80  C at a heating rate of 10  C min1. In the curve of the second heating process, the glass transition temperature (Tg) was determined from the endothermic stepwise change in the DSC heat flow. And the melting temperature (Tm) was read off from the corresponding peak. 2.5. Preparation and characterization of Alkyl-PEI/SPIO nanovehicles Detailed synthetic method for SPIO nanoparticles had been described in a previous publication [22]. These nanocrystals in hexane were dried under argon and redispersed in chloroform together with Alkyl-PEI. Then, mixed solution was slowly added into water with sonication. The mixture was under shaking overnight and the remaining chloroform was removed through rotary evaporation. The nanovehicles were characterized through dynamic light scattering (DLS) and zeta-potential. The content of Alkyl-PEI and SPIO nanoparticles in the final formulations were determined by thermo gravimetric analysis (TGA) using a Netzsch STA 449C Jupiter instrument (Germany). The T2 relaxivity of nanovehicles was measured at 1.5T on a clinical MR scanner (Siemens, Germany) at room temperature as described [19]. 2.6. Preparation and characterization of Alkyl-PEI/SPIO/siRNA nanocomplexes Following the reported method [18], the Alkyl-PEI/SPIO/siRNA nanocomplexes with different PEI nitrogen/nucleic acid phosphate (N/P) ratios (N/P ratios from 1 to 40) were prepared by mixing an appropriate amount of Alkyl-PEI/SPIO nanovehicles with siRNA in PBS. The obtained nanocomplexes were incubated at room temperature for 20 min. The Alkyl-PEI/SPIO/siRNA nanocomplexes were characterized by DLS and zeta-potential. 2.7. Agarose gel electrophoresis analysis For gel retardation assay, Alkyl-PEI/SPIO/siRNA nanocomplexes were incubated at room temperature (RT) for 20 min, and then nanocomplexes were loaded onto 1% (w/v) agarose gel containing 1% (v/v) GoodView with tris-acetate-EDTA (TAE) running buffering. Gel electrophoresis was carried out at 100 V for 15 min and the gel was subsequently imaged using a Bio-rad Gel Doc XR System (USA). The binding capacity was expressed by the threshold N/P ratio, above which siRNA failed to enter the gel. For heparin decomplexation assay, Alkyl-PEI/SPIO/siRNA nanocomplexes (N/ P ¼ 20) were prepared as described above. Various amounts of heparin were added and the mixtures were incubated for 15 min. And the samples were loaded on a 1% agarose gel and electrophoresis was carried out as described above. For serum stability assay, the Alkyl-PEI/SPIO nanovehicles were incubated with siRNA for 20 min. After adding serum at a 50% concentration, Alkyl-PEI/SPIO/siRNA nanocomplexes were incubated for predetermined periods at 37  C. The resulting samples were treated with 10 mg heparin for 15 min, then loaded on a 1% agarose gel and subjected to electrophoresis as described above. 2.8. Cell viability assays of Alkyl-PEI/SPIO/siRNA nanocomplexes The evaluation of cytotoxicity of Alkyl-PEI/SPIO/siRNA nanocomplexes with different siRNA concentrations was performed using a DNA assay based on Hoechst 33258 staining. RAW264.7 cells were seeded onto 48-well plates at a density of 1  104 cells/well and incubated for 24 h. Alkyl-PEI/SPIO/siRNA nanocomplexes were added and incubated with the cells for 12 h. Then the medium was replaced with 500 mL fresh medium and cells were incubated in a CO2 incubator for another 48 h. Control cells were treated with fresh medium only. Cytotoxicities were evaluated by using Hoechst 33258 staining assay, 48 h posttransfection. 2.9. Internalization of Alkyl-PEI/SPIO/siRNA nanocomplexes Confocal laser scanning microscope (CLSM) was used to assess the intracellular trafficking of Alkyl-PEI/SPIO/siRNA nanocomplexes. MCF-7/ADR cells were seeded on 35 mm glass-bottom dishes at a density of 1  105 cells/mL and incubated for 24 h in a CO2 incubator. Then cells were incubated with FITC labeled Alkyl-PEI/SPIO/ siRNA nanocomplexes (N/P ¼ 20) for 12 h, and the concentration of siRNA was 50 nM. After that the medium was removed, and cells were washed with PBS and fixed with 4% formaldehyde for 10 min at RT, and washed with PBS. And cell nuclei were stained with DAPI for 5 min at RT and washed with PBS. Under confocal laser scanning microscopy (CLSM; TCS SP4, Leica Microsystems, Germany), the images were observed and captured.

2.3. Critical micelle concentration (CMC) CMC of Alkyl-PEIs was estimated by the standard pyrene method on a Hitachi F7000 fluorescence spectrometer (Japan) [21]. Briefly, a known amount of pyrene in acetone was added to vials and then evaporated. Aqueous micelle solutions at varied concentrations were added to each vial, and the vial was under shaking overnight. The final concentration of pyrene was fixed at 6.0  107 M. The excitation wavelength was adjusted to 395 nm, and the fluorescence intensity at 338 and 334 nm were monitored. The ratios between intensities at 338 nm and 334 nm (intensity ratio, I338/I334) were determined for each sample.

2.10. In vitro cell transfection MCF-7/ADR cells were seeded onto 6-well plates at a density of 2  105 cells/well and incubated for 24 h. Transfection complexes were prepared as follows: siRNA (50 nM, 100 pmol per well) and appropriate amount of Alkyl-PEI/SPIO were both diluted to 250 mL with blank medium for 5 min. After that, the solutions were mixed and incubated at RT for another 20 min. The transfection complexes or transfection agents alone were then added to the wells and the cells were incubated for 12 h. Then the medium was replaced with fresh medium and cells were cultured for

G. Lin et al. / Biomaterials 35 (2014) 9495e9507

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siRNA loading

Alkyl-PEI/SPIO nanovehicles

Alkyl-PEI/SPIO/siRNA nanocomplexes

P-glycoprotein (P-gp)

Extracellular matrix

P-gp downregulation

End

ocy

tos

Target mRNA degradation is

MR imaging Target mRNA inhibition

SC ly RI mb se as

En d es oso ca ma pe l

siRNA release

Cytoplasm

Nucleus

Scheme 1. Schematic representation of Alkyl-PEI/SPIO/P-gp-siRNA nanocomplexes for P-gp silencing. First, nanocomplexes of P-gp-siRNA and Alkyl-PEI/SPIO were formed through electrostatic interaction. Internalization of Alkyl-PEI/SPIO/P-gp-siRNA nanocomplexes was realized by a non-receptor mediated endocytosis. Endosomal escape was required for the nanocomplexes to avoid siRNA degradation. P-gp-siRNA was released from the nanocomplexes and unwinded by the RNA-induced silencing complex (RISC), leading to the inhibition of the target mRNA. Finally, the target mRNA was degraded and the expression of P-gp was downregulated. another 48 h. Cells were then harvested for immunoblotting. Cells transfected with P-gp-siRNA by Lipofectamine 2000 served as a positive control and untransfected cells were used as a negative control. Protein abundance was quantified by ImageJ2x software (2.1.4.7, Rawak Software, Inc).

For a further investigation of transfection efficiency, two formulations (PEI-12C0.296/SPIO and PEI-18C-0.262/SPIO) were chosen from the results of western blot and immufluorescence analysis was performed according to the reported protocol [23]. MCF-7/ADR cells were seeded on 35 mm glass-bottom dishes at a density of

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1  105 cells/dish and maintained in a CO2 incubator. Twenty-four hours later, cells were transfected as described above and siRNA concentration was 50 nM. After 48 h, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at RT. Then cells were washed 5 min for 3 times on a shaker at 50 rpm and were blocked with blocking buffer for 1 h on a shaker at 50 rpm at RT. Cells were washed 5 min for 3 times on a shaker at 50 rpm and incubated with primary antibody anti-MDR1 (1:200) on a shaker at 50 rpm at 4  C overnight. After that, cells were washed 5 min for 3 times and incubated with secondary antibody Cy5-goat-anti-mouse (1:200) for 1 h. Then cells were washed 5 min for 3 times on a shaker at 50 rpm. And the nuclei were stained with DAPI for 10 min on a shaker at 50 rpm. Then cells were washed 5 min for 3 times, and 1.5 mL PBS were added into dishes. Under confocal laser scanning microscopy (CLSM; TCS SP4, Leica Microsystems, Germany), the images were observed and captured.

CMC (μM)

1000

Octadecyl Dododecyl Hexacyl *

100

10

2.11. Cytotoxicity studies of doxorubicin after P-gp silencing The effect of P-gp silencing on Dox toxicity was studied in MCF-7/ADR cells. Cells were seeded on 48-well plates at a density of 2  104 cells/well. After 24 h, cells were treated with Alkyl-PEI/SPIO/P-gp-siRNA nanocomplexes for 12 h. The medium was removed, and cells were further incubated with fresh medium for another 48 h. Then cells were incubated with Dox for another 24 h. Control cells were treated with Dox only. Cell viability was measured using the CCK-8 assay. The absorbance at 450 nm were measured using a Thermo scientific microplate reader.

1 6

2.12. MRI study of transfected cells MRI study of transfected cells was done following a previous publication [18]. At the end of transfection, cells were washed three times with PBS and harvested. Cells were dispersed in gelatin (1%) crosslinked with glutaraldehyde inside microcentrifuge tubes for MRI analysis. T2 relaxation measurements were performed on a Siemens 3T clinical MRI scanner (Germany) with a 5 cm volume coil and using spinecho imaging sequences. Images were acquired using a repetition time (TR) of 5000 ms and an echo time (TE) of 20 ms. The spatial resolution parameters were as follows: acquisition matrix of 250  191 mm, section thickness of 2 mm and two averages. The signal intensities of each sample were obtained through an analysis program provided by the MRI scanner. And the signal intensity of transfected cells was displayed as percentiles relative to untreated cells.

18 30 12 24 Degree of Alkylation (%)

36

Fig. 1. CMC values of Alkyl-PEIs (Determined using pyrene as a fluorescence probe (mean ± S. D., n ¼ 3)). * The CMC value of PEI-6C-0.095 was not determined.

comparison of thermal properties of PEI and Alkyl-PEIs samples, due to the same history of thermal treatment for all the samples in DSC measurement. PEI2k was an amorphous polymer with a Tg of 53.7  C. With increasing degree of alkylation, Tg increased gradually (Table 1). It might be attributed to the substitution group in the branch which made the polymer chain segment more rigid. It was noticed that PEI-18C-0.099 exhibited two Tgs (one at 44.2  C, the other at 4.2  C). This phenomenon might be contributed to the occurrence of microphase separation in PEI18C-0.099 [24]. It was noticed that none of the PEI-6Cs showed a Tm in the temperature ranging from 80 to 80  C (Table 1). This phenomenon was in consistence with the previous report that the side chain crystallization behavior could be observed when the number of carbon atoms in the side chains was 12 or higher [25e27]. Endothermic or exothermic transitions were found for all the PEI-12Cs and PEI-18Cs copolymers, and the Tm of Alkyl-PEI gradually increased with the higher degree of alkylation (Table 1). This might be assigned to the crystallization of the side chains which became stronger with increasing degree of alkylation [28,29]. It was found that Tm values of PEI-12Cs or PEI-18Cs with high degree of alkylation were slight higher than those of corresponding n-alkanes. It might be

2.13. In vivo transfection All studies involving animals were approved by the institute's animal care and use committee. Orthotopic tumor models were prepared by injection of 1  106 MCF-7/ADR cells suspended in 50 mL Matrigel (BD) into the breast of female nude mice (6e8 week old). When the diameter of tumors reached about 4 mm, 250 pmol of P-gp-siRNA complexed with Alkyl-PEI/SPIO at an N/P ratio of 20 were intratumorally injected every other two days for 4 times. The control groups were injected with an equal volume of PBS. At the end of experiments, each tumor tissue were excised and grounded in liquid nitrogen. Then the tissue powder was lysed in lysis buffer with sonication. The relative protein abundance across samples was determined for western blot.

3. Results and discussion 3.1. Thermal behavior of Alkyl-PEIs The thermal properties of Alkyl-PEIs were investigated by DSC. The second heating process was used for evaluation and Table 1 Structural parameters of Alkyl-PEIs and their physicochemical properties. Elemental analysis

DSC analysis

Polymer

Substitution groups

DA (%)

Mw

CMC (mM)

Tg ( C)

Tm ( C)

DHm (Jg1)

DSm (Jg1 K1)

PEI-6C-0.095 PEI-6C-0.154 PEI-6C-0.197 PEI-6C-0.273 PEI-12C-0.090 PEI-12C-0.147 PEI-12C-0.210 PEI-12C-0.296 PEI-18C-0.099 PEI-18C-0.139 PEI-18C-0.196 PEI-18C-0.262

n-hexacyl n-hexacyl n-hexacyl n-hexacyl n-dodecyl n-dodecyl n-dodecyl n-dodecyl n-octadecyl n-octadecyl n-octadecyl n-octadecyl

9.5 15.4 19.7 27.3 9.0 14.7 21.0 29.6 9.9 13.9 19.6 26.2

2123 2348 2500 2760 2437 2834 3277 3894 2853 3266 3868 4575

N/A 460 384 156 86.6 40.5 16.5 8.6 23.7 20.4 17.2 13.6

52.7 40.1 35.6 34.1 51.8 46.3 33.3 31.6 44.2/4.2c 42.0 41.3 31.1

N/A N/A N/A N/A 20.7 7.4 10.5 10.3 42.9 46.1 49.5 46.3

N/A N/A N/A N/A 3.9 23.4 28.7 30.4 17.4 40.6 44.8 66.4

N/A N/A N/A N/A 0.016 0.088 0.109 0.116 0.055 0.127 0.139 0.208

a b c

a

DA represented degree of alkylation relative to the total amino groups of PEI. Determined using pyrene as a fluorescence probe. Two Tgs were observed in PEI-18C-0.099 samples.

b

G. Lin et al. / Biomaterials 35 (2014) 9495e9507

contributed to the difference between the packing mode of the side chains in Alkyl-PEI and that of the corresponding n-alkanes [29]. Table 1 also showed the derivative with higher degree of alkylation had higher melting enthalpies (DHm). It was considered

30

that the increased degree of alkylation might increase the crystallinity of the amphiphilic polymers [30]. And it was found that the values of melt entropies (DSm) also increased with increasing the degree of alkylation. This was consistent with a previous report that

B

SPIO in hexane

25

15 10 5 0 1

10 Diameter (nm)

D

10 5 1000

100 Diameter (nm)

130

Diameter Zeta-potential

30

120

PEI-18C-0.262/SPIO

Diameter (nm)

25

15

0 10

100

C

20 Number (%)

PEI-12C-0.296/SPIO

20

20

Number (%)

Number (%)

25

15 10

20 110 10 100 0 90 -10

5

80

0 10

70

Zeta-potential (mV)

A

9499

-20

E

Diameter Zeta-potential

10 20 N/P ratio

F

40

PEI-12C-0.296/SPIO

30 20 10

120

0 110

-10 -20

100

240

Zeta-potential (mV)

130

90

5

300

140

Diameter (nm)

1000

100 Diameter (nm)

10 20 N/P ratio

y = 596.4x + 3.9

180 120

y = 515.3x + 4.8

60 0

-30 5

PEI-18C-0.262/SPIO

0

40

G

0.12 0.24 0.36 0.48 Iron concentration (mM)

Iron Concentration (mM) Water

0.03

0.06

0.10

0.15

0.25

0.30

0.40

PEI-12C-0.296/SPIO

PEI-18C-0.262/SPIO Fig. 2. Characterization of the SPIO nanocrystals and PEI-12C-0.296/SPIO, PEI-18C-0.262/SPIO nanovehicles and nanocomplexes. (A) Diameter of SPIO nanoparticles in hexane; (B) diameter of PEI-12C-0.296/SPIO nanovehicles in water; (C) diameter of PEI-18C-0.262/SPIO nanovehicles in water; (D) diameter and zeta-potential of PEI-12C-0.296/SPIO/siRNA nanocomplexes at various N/P ratios; (E) diameter and zeta-potential of PEI-18C-0.262/SPIO/siRNA nanocomplexes at various N/P ratios; (F) T2 relaxation rate (1/T2, s1) as a function of Fe concentration (mM) for PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles at 1.5T; (G) T2-weighted MRI images (1.5T, spin-echo sequence: TR ¼ 5000 ms, TE ¼ 18 ms) of PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles.

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the ordered degree of molecular conformation and packing in crystalline region improved with increased degree of alkylation [29].

containing vehicles [33]. T2-weighted signal intensity decreased gradually when the Fe concentration increased (Fig. 2G). 3.4. Agarose gel electrophoresis analysis

3.2. Critical micelle concentration (CMC) To select stable polymer nanovehicles, CMC values were determined by a steady-state fluorescent spectroscopy study. Although there were several methods to determine CMC value for amphiphilic polymers, the fluorescent spectroscopy was sensitive enough and easy to use [21]. The CMC value was determined from the onset of an increase in the intensity ratio I338/I334 plotted versus the logarithm of polymer concentrations. It was found that as the alkylation chain length increased, the value of CMC decreased gradually. Moreover, with an increased degree of alkylation, CMC value also decreased accordingly (Fig. 1, Table 1). It was in good agreement with a previous report that increasing hydrophobic segments of amphiphilic polymer to a certain degree would facilitate micellization and produce more stable micelles [31]. The CMC values of PEI-6Cs were in the range of 0.3e1.5 mg/mL, corresponding to values of 1  104 to 5  104 M. These values were much higher than the CMC values reported for amphiphilic PEI2k which were able to form stable micelles, suggesting poor stability of micelles from PEI-6Cs [31]. In contrast, CMC values of PEI-12Cs and PEI-18Cs (9  106 to 5  105 M) were much lower than that of PEI6Cs, and close to the reported amphiphilic PEI2k CMC values. PEI12Cs and PEI-18Cs samples were expected to form more thermodynamically stable micelles that were able to maintain the integrity upon dilution. From the results of CMC, five amphiphilic PEI (PEI12C-0.210, PEI-12C-0.296, PEI-18C-0.139, PEI-18C-0.196, PEI-18C0.262) were chosen to form the Alkyl-PEI/SPIO nanovehicles because they had relatively low CMC values. 3.3. Self-assembly of Alkyl-PEI/SPIO nanovehicles As shown in Fig. 2A, the monodispersed SPIO nanoparticles had a narrow size distribution of 10.4 ± 2.7 nm. DLS analyses (Fig. 2B, C) showed that PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles were monodispersed without obvious aggregation. The sizes of Alkyl-PEI/SPIO nanovehicles were ranging from 80 to 130 nm (Table 2). The averaged diameter of the PEI-12C-0.296/ SPIO/siRNA and PEI-18C-0.262/SPIO/siRNA nanocomplexes in water were in the range of 80e130 nm (Fig. 2D, E). And the diameters of PEI-12C-0.296/SPIO/siRNA and PEI-18C-0.262/SPIO/siRNA nanocomplexes were close to that of corresponding nanovehicles. Although the bound siRNA can alter the diameter of nanocomplexes in a certain degree, the size of PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO played an essential role in determining the diameter of the resulting nanocomplexes [18]. The zeta-potential results showed that the nanovehicles were positively charged, with a potential ranging from þ36 mV to þ44 mV (Table 2), due to the presence of multiple amino groups in the Alkyl-PEI. A high charge density would contribute to a good stability of nanovehicles in water [32]. In fact, the nanovehicles were found to be stable in water for 12 months. The zeta-potentials of the PEI-12C-0.296/SPIO/siRNA and PEI-18C-0.262/SPIO/siRNA would decrease when the N/P ratio decreased (Fig. 2D, E), indicating the successful loading of siRNA. To understand the MR imaging capacity of PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles, their relaxivity studies were carried out under a 1.5T clinical MRI scanner. The r2 value of PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles were 514.7 Fe mM1 s1 and 596.8 Fe mM1 s1 respectively (Fig. 2F), and these values were much higher compared with single particle

Agarose gel electrophoresis was performed to evaluate the binding ability of siRNA to Alkyl-PEI/SPIO nanovehicles. The agarose gel electrophoresis assay revealed that Alkyl-PEI/SPIO nanovehicles with higher degree of substitution possessed lower binding ability (Fig. 3A, Table 2). In general, primary amino groups were known to condense siRNA better than other forms of amines, due to their higher protonation degree at a given pH [34]. Primary amino groups were alkylated preferentially in this synthetic route for alkylation [20]. So the percentage of primary amino groups of Alkyl-PEI decreased with an increased degree of alkylation. Interestingly, PEI-18C-0.196/SPIO had a best binding ability in PEI-18C/ SPIO group. The binding affinity was improved with an increase in degree of substitution, but compromised with further increasing degree of substitution, and similar findings were reported recently [35]. It was suggested that a minor difference in nanovehicle structures could have significant impact on binding affinity. In a heparin decomplexation assay, siRNA was found released from Alkyl-PEI/SPIO/siRNA nanocomplexes when heparin was added (Fig. 3B), presumably due to the latter's stronger competitive interaction with the particle surface [36]. Due to rapid degradation of siRNA in plasma and cellular cytoplasm, a qualified nucleic acid delivery nanovehicle should be able to protect them from attacking by enzymes. The results showed naked siRNA was degraded rapidly within 6 h when incubated with 50% FBS at 37  C. In contrast, it could survive for 24 h, and even for 48 h when complexed with Alkyl-PEI/SPIO nanovehicles (Fig. 3C). These results demonstrated that Alkyl-PEI/SPIO nanovehicles could effectively protect siRNA from RNase degradation. 3.5. Cytotoxicity and internalization of Alkyl-PEI/SPIO/siRNA nanocomplexes Cytotoxicity became a primary concern for cationic polymeric gene delivery including PEI25k. It was reported that it could induce severe cytotoxicity through non-specific interactions with negatively charged biomolecules [37e39]. However, it was considered that PEI2k was relatively safer in cell labeling and gene transfection [40]. The Hoechst 33258 staining assay was used to investigate the cytotoxicity of Alkyl-PEI/SPIO/siRNA nanocomplexes on RAW264.7 cells. As shown in Fig. 4A, the Alkyl-PEI/SPIO/siRNA nanocomplexes had no obvious cytotoxicity on RAW264.7 cells within the transfection concentrations (100 nM), which was consistent with the previous report about Alkyl-PEI/SPIO nanovehicles [18,41]. However, when the concentration of siRNA increased to 200 nM, viability of cell line treated with PEI-18C-0.262/SPIO/siRNA decreased to 88% Table 2 Characterization of Alkyl-PEI/SPIO nanovehicles Polymer

DAa (%)

Polymer : SPIOb

Sizec (nm)

PEI-12C-0.210 PEI-12C-0.296 PEI-18C-0.139 PEI-18C-0.196 PEI-18C-0.262

21.0 29.6 13.9 19.6 26.2

0.6 0.6 0.6 0.6 0.6

87.4 99.8 99.4 92.8 124.7

a b c d

± ± ± ± ±

6.8 4.4 3.0 3.0 6.6

Zetac (mV)

PDIc

Complete siRNA boundd (N/P)

± ± ± ± ±

0.31 0.30 0.26 0.33 0.28

10 20 10 5 20

43.8 39.3 42.2 43.8 36.9

0.4 0.6 0.9 0.4 0.7

Determined by elemental analysis. Mass ratio. Determined by dynamic light scattering (mean ± S. D., n ¼ 3). Determined by agarose gel electrophoresis.

G. Lin et al. / Biomaterials 35 (2014) 9495e9507

To investigate the details of intracellular location of the AlkylPEI/SPIO/siRNA nanocomplexes, FITC was used to label Alkyl-PEI. The nuclei of cells were stained with DAPI. The CLSM images were shown in Fig. 4B. The images revealed that almost all Alkyl-

from 95% at 100 nM. The minor cytotoxicity of PEI-18C-0.262/SPIO might be attributed to the high alkylation degree which strengthened the interaction between nanovehicles with cell membrane, ultimately leading to the disruption of the plasma membrane [42].

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Fig. 3. Agarose gel electrophoresis analysis of Alkyl-PEI/SPIO/siRNA nanocomplexes: (A) Electrophoretic retardation analysis of siRNA binding with Alkyl-PEI/SPIO nanovehicles; (B) release of siRNA after the addition of heparin at various weight ratios; (C) serum stability of siRNA when complexed with Alkyl-PEI/SPIO at an N:P ratio of 20; the study was performed in 50% serum solution for a predetermined incubation time. The arrows in (A) represented complete siRNA complexation N/P threshold, and the one in (C) represented the time point at which siRNA remained.

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A

PEI-12C-0.210/SPIO/siRNA PEI-12C-0.296/SPIO/siRNA PEI-18C-0.139/SPIO/siRNA PEI-18C-0.196/SPIO/siRNA PEI-18C-0.262/SPIO/siRNA

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Fig. 4. Cytoxicity and cellular uptake of Alkyl-PEI/SPIO/siRNA nanocomplexes. (A) Cell viabilities of RAW264.7 cells with different concentrations siRNA complexed with Alkyl-PEI/ SPIO at the N/P ratio of 20. The data were expressed as mean values (with standard deviation) of three experiments; (B) cellular uptake of nanocomplexes by MCF-7/ADR cells: confocal microscopic images of MCF-7/ADR cells after transfection with FITC labeled PEI-12C-0.296/SPIO/siRNA; the nuclei were stained with DAPI and the internalized nanocomplexes appeared green in the fluorescence images (scale bar: 25 mm).

PEI2k/SPIO/siRNA nanocomplexes were localized in the cytoplasm. This observation was different from the cellular distribution of unmodified PEI/siRNA complexes, who localized not only in the cytoplasm but also in the nuclei [43,44]. Thus Alkyl-PEI2k/SPIO nanovehicles can alter the intracellular distribution of siRNA, and such alteration may bring an enhancement in gene silencing efficiency [18]. 3.6. In vitro cell transfection Western blot assay was used to assess the efficacy of P-gp-siRNA delivered by Alkyl-PEI/SPIO nanovehicles and knockdown the expression of P-gp. The N/P ratio was 20 in the case of Alkyl-PEI/ SPIO as it was found to be optimal in previous publications [18,20]. No P-gp knockdown was observed in the free P-gp-siRNA or Alkyl-PEI/SPIO alone groups. Meanwhile, it was demonstrated that Alkyl-PEI/SPIO nanovehicles could effectively deliver P-gp-siRNA into MCF-7/ADR cells and knockdown the expression of P-gp (Fig. 5A). Nanovehicles with higher degree of alkylation possessed higher P-gp silencing efficiency than that of lower ones. The possible reason may lie in that higher degree of long alkyl

substituent on PEI enabled stronger interaction of Alkyl-PEI/SPIO/ siRNA nanocomplexes with cell membrane, hence to promote endocytosis of the nanocomplexes, and in turn, to bring a higher Pgp silencing efficiency [20,45]. PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles produced effective P-gp silencing efficacy (50e60%) comparable to that of some non-viral gene delivery in previous publications [46,47]. However, most previous studies for modulation of P-gp expression were performed at siRNA concentrations above 100 nM, and such high siRNA dose may cause non-specific effects [48]. Thus P-gp downregulation using siRNA at 50 nM by PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles may reduce non-specific effects and enhance the utility of siRNA. PEI-12C-0.296/SPIO and PEI-18C0.262/SPIO were chosen for further studies, due to their excellent transfection efficacy. Immunofluorescence staining was used to detect the expression of P-gp. As identified by Cy5, P-gp was overexpressed in untransfected MCF-7/ADR cells. To investigate the effect of PEI-12C-0.296/ SPIO/P-gp-siRNA and PEI-18C-0.262/SPIO/P-gp-siRNA nanocompl exes on the expression of P-gp, MCF-7/ADR cells were treated with the two chosen siRNA formulations. As shown in Fig. 5B, P-gp

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Fig. 5. Detection of P-gp expression. (A) Detection of P-gp knockdown in MCF-7/ADR cells by Alkyl-PEI/SPIO/P-gp-siRNA using western blot. Untransfected cells were used as a negative control and transfected cells with P-gp-siRNA by Lipofectamine 2000 were used as a positive control. The relevant P-gp expression was calculated by the signal intensity of the protein bands. PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO were chosen for further studies, due to their excellent transfection efficacy. (B) Immunofluorescence staining for P-gp in untransfected MCF-7/ADR cells and cells that had been transfected with P-gp-siRNA complexed with PEI-12C-0.296/SPIO or PEI-18C-0.262/SPIO nanovehicles, respectively. Untransfected cells were used as a control. Indirect immunostaining was performed using primary antibody anti-MDR1 and secondary antibody Cy5-goat-anti-mouse (a), cell nuclei were stained with DAPI (b), and it was merged with (a) to detect intracellular distribution of P-gp (c).

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Cell Viability (%)

3.7. Cytotoxicity studies after P-gp silencing

Doxorubicin alone PEI-12C/SPIO PEI-12C/SPIO/P-gp-siRNA PEI-18C/SPIO PEI-18C/SPIO/P-gp-siRNA

To investigate whether the therapeutic efficacy of anticancer drug doxorubicin on MCF-7/ADR cells will improve after the Pgp silencing, cytotoxicity studies were performed using CCK-8 assay. As shown in Fig. 6, there was no significant difference between Alkyl-PEI/SPIO and Dox alone group. However, when the cells were treated with PEI-12C-0.296/SPIO/P-gp-siRNA or PEI-18C-0.262/SPIO/P-gp-siRNA nanocomplexes, cell viability decreased significantly comparing to Dox alone group. At a Dox concentration of 5 mML, cell viability of the drug-only control group was 86%. In contrast, cells treated with nanovehicle/P-gpsiRNA formulations had cell viability of 63% and 59%, respectively for PEI-12C-0.296/SPIO/P-gp-siRNA and PEI-18C-0.262/ SPIO/P-gp-siRNA nanocomposites. And when the Dox concentration increased to 40 mM, the viability of nanovehicle/P-gpsiRNA formulations treated cells dropped to 15% and 13% compared with 55% for the control group. These results clearly showed that P-gp downregulation by nanocomplexes can reverse the MDR through inhibiting the drug efflux activity of the transporter, and resulted in an increase in drug sensitivity on MCF-7/ADR cells [8,49].

80

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0 0

10 40 20 30 Doxorubicin concentration (μM)

Fig. 6. Doxorubicin cytotoxicity in MCF-7/ADR cells after transfected with Alkyl-PEI/ SPIO or Alkyl-PEI/SPIO/siRNA nanocomplexes. MCF-7/ADR cells were treated with formulations with or without P-gp-siRNA. Control cells were treated with the medium only. After 12 h, the medium was refreshed. Cells were reincubated for 48 h. Then cells were treated with doxorubicin at different concentrations for 24 h and the viability was measured. Data were expressed as the mean ± standard deviation (n ¼ 3). The alkylation degree of the corresponding polymer in PEI-12C/SPIO and PEI-18C/SPIO were 29.6% and 26.2%, respectively. There is a significant difference for Alkyl-PEI/ SPIO/siRNA treatments versus drug only treated cells for all concentration-points in MCF-7/ADR cells (p < 0.05).

3.8. MRI study of transfected cells With the help of functional contrast agents, MRI is an excellent tool to track the delivered genes during a therapy [18,50]. In our study, r2 value of the PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles were 514.7 Fe mM1 s1 and 596.8 Fe mM1 s1 respectively (Fig. 2G). In general, a T2 MRI imaging agent with better contrast effect would display a higher r2 value. The r2 values of our nanovehicles were much higher than that of a commercial MRI T2 contrast agents, such as Feridex [51]. Under a clinical 3T MRI, the transfected cells exhibited a significantly decreased signal intensity (75.2% and 84.9% for PEI-12C-0.296/SPIO and PEI-18C0.262/SPIO, respectively) compared with the control (Fig. 7). SPIO-based MRI contrast agents could shorten the T2 (spinespin)

expression in MCF-7/ADR cells that were treated with siRNA formulations decreased significantly compared with the control. The results were in accordence with western blot assay, suggesting Pgp-siRNA that was complexed with either PEI-12C-0.296/SPIO or PEI-18C-0.262/SPIO could reduce expression of P-gp significantly.

A

B

120

*

* Water

PEI-12C/SPIO/siRNA

PEI-18C/SPIO/siRNA

Relative signal intensity (%)

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100 80 60 40 20

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PE

PE

I-1

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Co

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ro

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Fig. 7. (A) T2-weighted magnetic resonance images of agarose gel phantoms containing the MCF-7/ADR cells treated with Alkyl-PEI/SPIO/siRNA nanocomplexes; (B) relative T2 signal intensity (3T, spin echo acquisition) of PEI-12C/SPIO/siRNA and PEI-18C/SPIO/siRNA nanocomplexes transfected cells (5  105 cells per tube) at N/P ratio of 20 in gelatin phantom in microcentrifuge tubes. *p < 0.001. The alkylation degree of the corresponding polymer in PEI-12C/SPIO and PEI-18C/SPIO were 29.6% and 26.2%, respectively.

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3.9. Therapeutic silencing effects in vivo

relaxation time. A higher r2 value contributed to the higher degree of decreased signal intensities of cells labeled by PEI-18C-0.262/ SPIO comparing to that of PEI-12C-0.296/SPIO group. It was demonstrated that our nanovehicles, PEI-12C-0.296/SPIO and PEI18C-0.262/SPIO, were suitable for MRI contrast enhancement [18,19].

To investigate the potential of Alkyl-PEI/SPIO for delivering of siRNA in MDR cancer therapy, the gene silencing ability was evaluated on a MCF-7/ADR animal model. Three groups of BALB/c mice (n ¼ 5/group) bearing orthotopic MCF-7/ADR tumors were

A

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Fig. 8. Detection of P-gp expression in tumor tissue. (A) Detection of P-gp knockdown by PEI-12C/SPIO/P-gp-siRNA and PEI-18C/SPIO/P-gp-siRNA using western blot. P-gp-siRNA complexed with PEI-12C/SPIO or PEI-18C/SPIO were intratumorally administered by four injections into the tumors (n ¼ 5). Tumors injected with PBS were used as a control. The relative P-gp expression was calculated by the signal intensity of the protein bands; (B) relative P-gp expression levels in MCF-7/ADR tumor treated with P-gp-siRNA complexed with PEI-12C/SPIO or PEI-18C/SPIO, compared with the PBS control (mean ± S. D., n ¼ 5). *p < 0.001. The alkylation degree of the corresponding polymer in PEI-12C/SPIO and PEI18C/SPIO were 29.6% and 26.2%, respectively.

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intratumorally injected with PBS, P-gp-siRNA complexed with PEI12C-0.296/SPIO or PEI-18C-0.262/SPIO nanovehicles. The nanocomplexes (250 pmol P-gp-siRNA in 50 mL PBS) or PBS (50 mL) were administered by four injections into different locations of tumors. Two days after the final injection, the tumors were excised and protein was extracted for western blot. Although many studies in cancer research employed a subcutaneous tumor model, the orthotopic one provides a more similar environment within the in situ natural development [52]. And in orthotopic models, the disease characterization could be better modeled. As shown in Fig. 8, local administration of P-gp-siRNA formulation induced significant reduction (71.4% and 62.9% for PEI12C-0.296/SPIO and PEI-18C-0.262/SPIO, respectively) in P-gp protein levels. These P-gp downregulation efficiency was much higher than that of unmodified PEI25k (29%) using a nearly same siRNA dose in a previous publication [53]. Taking the notorious toxixity of PEI25k into account, our nanovehicles (PEI-12C-0.296/ SPIO or PEI-18C-0.262/SPIO) were relatively better for P-gp-siRNA delivery to overcome MDR. In this study, we chose the local delivery method to introduce siRNA into the tumor tissue instead of intravenous systemic delivery. The Alkyl-PEI/SPIO/siRNA nanocomplexes were positively charged and it may bind to serum proteins nonspecifically, leading to unwanted aggregation and subsequent side effects. Conjugation of PEG on Alkyl-PEI may be helpful to reduce non-specific protein binding and increase the blood circulation time of nanocomplexes, leading to a better tumor targeting efficiency. We have synthesized a series of PEG modified Alkyl-PEI and will test their siRNA binding, transfection and in vivo delivery efficacy in our further studies. 4. Conclusions A series of amphiphilic Alkyl-PEI2k were synthesized and characterized. Dodecyl and octadecyl PEI were able to form stable nanocomposites with hydrophobic SPIO nanoparticles. These nanovehicles were suitable for siRNA delivery as they could bind siRNA efficiently and protect them from RNase degradation. Moreover, higher alkylation degree of PEI was helpful for better Pgp downregulation. PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO significantly enhanced gene silencing for P-gp in MCF-7/ADR cells, followed by improvement of the therapeutic efficacy of an anticancer drug doxorubicin. And the two formulations showed a high T2 relaxivity and transfected cells displayed high signal contrast compared with untreated ones. More importantly, the Pgp silencing capacity of the two formulations had been confirmed in a MCF-7/ADR orthotopic tumor model. Altogether, all these results demonstrated the potential of the MRI-visible nanovehicles, PEI-12C-0.296/SPIO and PEI-18C-0.262/SPIO nanovehicles, for siRNA delivery aimed at reversing MDR in cancer therapy. Acknowledgments The work was supported by National Key Basic Research Program of China (2013CB933903), National Key Technology R&D Program (2012BAI23B08), National Natural Science Foundation of China (20974065, 51173117 and 50830107), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, Grant No. IRT1272) of China. References [1] Gottesman MM. Mechanisms of cancer drug resistance. Annu Rev Med 2002;53:615e27. cs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting [2] Szaka multidrug resistance in cancer. Nat Rev Drug Discov 2006;5(3):219e34.

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