Nano Res.

Electronic Supplementary Material

Efficiency against multidrug resistance by co-delivery of doxorubicin and curcumin with a legumain-sensitive nanocarrier

Sen Lin1,2,3 (), Peiling Xie1, Mengmeng Luo1, Qing Li2,3, Ling Li1, Jinzhao Zhang1, Qinxiang Zheng1,

Hao Chen1,3, and Kaihui Nan1,3 ()

1 School of Ophthalmology & Optometry and Eye Hospital, Wenzhou Medical University, Wenzhou 325027, China 2 Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Science, Wenzhou 325000, China 3 School of Biomedical and Engineering, Wenzhou Medical University, Wenzhou 325027, China

Supporting information to https://doi.org/10.1007/s12274-017-1928-1

Materials

Chitosan (CS, 120 kDa), 4-dimethylaminopyridine (DMAP), 1-ethyl-3,3-dimethyl- aminopropyl carbodiimide

(EDC), N,N-diisopropyl-ethylamine (DIPEA), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate (HATU), and triisopropylsilane (TIS) were purchased from Sigma-Aldrich (St. Louis,

MO).  Doxorubicin (DOX), curcumin (Cur), trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), and

dichloromethane (DCM) were provided by Aladdin Industrial Co. Ltd. (Shanghai, China). Genipin, cyclohexane,

i-octanol, and triton X-100 were purchased from Shanghai Yare Bitech, Ltd., Co. (Shanghai, China). The legumain

substrate tetrapeptide N-(9-Fluorenylmethoxycarbonyl)-β-alanyl-L-alanyl-L-(N-γ-trityl-asparaginyl)-L-leucyl,

Fmoc-PEP(Trt)-OH) was purchased from Kangbei Ltd., Co (Ningbo, China). Recombined human legumain

protease was purchased from Novoprotein Scientific CO., (New Jersey, US). MCF-7 and MCF-7/ADR cell line

were purchased from American Type Culture Collection (ATCC, Manassas, VA). All reagents were analytical

grade.

Methods for DOX-PEP(Trt)-CH2-CH2-COOH synthesis

A method previously reported by Fernandez et al. 1 was employed to synthesize DOX-PEP(Trt)-CH2-CH2-COOH.

Briefly, DOX hydrochloride and Fmoc-AAN(Trt)L-OH were dissolved in DMF by ultrasound treatment.

DIPEA and HATU were added to initiate the reaction and the mixture was left for 2 h at room temperature

under stirring. The product was harvested by precipitation (cold water, 4 °C), centrifugation (10000 rpm, 10 min),

washing (acetate buffer, pH 4.0), and freeze drying. The resultant powder was further subjected to de-Fmoc

reaction with treatment of 20 % piperidin in DMF. After reaction at room temperature for 5 min, the mixture

was quickly cooled to -5 °C in a cold bath. Succinic anhydride was immediately added and the mixture was left

Address correspondence to Sen Lin, youhulinsen@hotmail.com; Kaihui Nan, nankh@163.com

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for 10 min in room temperature under stirring. The mixture was dialyzed against distilled water for 24 h using a

dialysis bag (Solarbio Life Sciences CO., Beijing, China) with a molecular weight cut off (MWCO) of 100–500 Da

to remove DMF, and further subjected to freeze drying to obtaining the crude product DOX-PEP(Trt)-

CH2-CH2-COOH. This crude product was purified using a chromatography column (1×50 cm) filled with

Sephadex LH-20 (GE Healthcare Bio-Sciences, Sweden). The column was eluted with CH3OH, and fractions

were collected within a regular interval. The collected fractions were further purified by semi-preparative high

performance liquid phase (Shimadzu Corporation, Kyoto, Japan) equipped with a PRC-ODS preparative

column (25×460 mm) using water (A) and methanol (B) as the mobile phase with the following composition: 0-5

min 20% B, 5-20 min 40%, 20-35 min 60%, 35-50 min 80% B. The chemical structures of the purified products

were analyzed by 1H-NMR and ESI-MS. NMR spectra were recorded by a Bruker DRX-600 spectrometer

(Bruker, Rheinstetten, Germany) in deuterated DMSO-d6 at room temperature. ESI-MS were obtained using

Shimadzu (Kyoto, Japan) LCMS 8050 triple quadrupole MS system equipped with a heated capillary interface

and electrospray ionization (ESI) source.

Methods for DOX-PEP-CH2-CH2-CONH-CS synthesis

DOX-PEP(Trt)-CH2-CH2-COOH was conjugated with CS amino group via chemical reaction in the presence of

EDC and DMAP.2 CS (161 mg, equal to 1 mM of amino group) was mixed with different amount of

DOX-PEP(Trt)-CH2-CH2-COOH in a MES buffer (pH 4.0). The mole ratio of amino group in CS/carboxyl group

in DOX-PEP(Trt)-CH2-CH2-COOH was defined as 5:1, followed by addition of 1.3 carboxyl group equivalent of

EDC and 0.1 DMAP, and left at room temperature for 48 h. The resultant mixture was dialyzed against distilled

water using a dialysis bag with a MWCO of 14000 Da. DOX-PEP(Trt)-CH2-CH2-CONH-CS powder was then

obtained after freeze drying. The conjugation rate was evaluated by fluorescence measurement with emission

wavelengths at 590 nm and excitation wavelengths at 470 nm. The measured fluorescence intensity was converted

into DOX amount according to the curve using free DOX as standard. The conjugation rate was calculated by

dividing the amount of measured DOX by the amount of DOX-PEP(Trt)-CH2-CH2-CONH-CS employed.

Methods for DOX-PEP-CH2-CH2-CONH-CS crosslinking

DOX-PEP-CH2-CH2-CONH-CS was crosslinked using a method previously described3 with some modifications.

Briefly, a mixture containing cyclohexane (18.15 mL), 1-octanol (4.41 mL), and triton X-100 (4.74 g) was prepared

and emulsified for 7 min using a homogenizer (IKA, T10) at a speed of 20000 rpm. A solution (0.1% acetic acid,

1.1 mL) containing 33 mg of DOX-PEP(Trt)-CH2-CH2-CONH-CS was added into the mixture and ultrasound

treated for 5 min. The crosslinking was initiated by adding 200 μL genipin (0.5 mM) in ethyl alcohol and left for

24 h at room temperature in the dark. The DOX-PEP(Trt)-CH2-CH2-CONH-CS NPs were harvested using the

following steps in sequence: precipitation (isopropyl alcohol), centrifugation (10000 rpm, 10 min), and lyophilization.

In order to be legumain responsive, this NP was subjected to de-Trt reaction using TFA, DCM, and TIS at the

ratio of 4.9:4.9:0.2 (v/v/v) for 1 h at room temperature. After evaporation under vacuum at 40 °C to remove DCM,

the resulting suspension was dialyzed against distilled water using a dialysis bag with a MWCO of 14000 Da.

The chemical structure was confirmed by Fourier transform–infrared (FT-IR) spectra.

Methods for drugs (Cur and DOX-Leu) determination by Ultra-high Performance Liquid Chromatography-mass-

mass (UPLC-MS-MS)

The amount of Cur was determined with a UPLC-MS-MS. A Shimadzu LC-8050 UPLC-MS-MS system equipped

with a Shim-pack XR-ODS III (2.0 mm i.d. × 75 mm, particle size: 1.6 μm) chromatography column and a triple

quadrupole mass spectrometer were employed for compound analysis. Samples (2 μL) were loaded on UPLC

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by an auto-sampler, and eluted at 40 °C. Solvent A (0.1% formic acid) and B (methanol) were used as mobile

phases at a flow rate of 0.4 mL/min, with the following elution: 0–0.5 min, 60% B; 4.5 min, 90% B; 5.5 min, 90% B;

and 6 min, 60% B. The UPLC system was interfaced with a triple quadrupole mass spectrometer fitted with an

APCI ionization source operating in Auto-MSn mode (smart fragmentation). The MS was operated in the

negative ionization mode with capillary voltage at 4 kV. The nebulizing and drying gas flow were set at 3 and

5 L/min respectively. The temperature of the interface, desolvation area, and heart block were set at 350 °C,

250 °C, and 250 °C respectively. Mass spectrometer was performed with multiple reaction monitoring (MRM)

mode. The peak area of m/z 367.4>134.1 was employed for Cur quantitative analysis.

With the action of legumain, a DOX-leucinyl deviate (DOX-Leu) was released. DOX-Leu was analyzed by

UPLC-MS-MS using the method described in our previously report4 with some modifications. The analysis was

conducted using the above-mentioned UPLC-MS-MS equipment. Due to the relative hydrophilic properties

compared to Cur, ESI ionization source instead of the APCI one was used for DOX-Leu detection. The

chromatographic condition was set as same as Cur analysis except for the following eluting conditions: 0-1 min,

5% B; 7.5, 90 % B, 8 min, 90 % B. The mass spectrometer was operated in the negative ionization mode with

capillary voltage at 4 kV. It was performed with Auto-MSn manner using a ramping of the collision energy in

MRM mode. The peak area of m/z 655.5>395.1 was employed for DOX-Leu quantitative analysis. The nebulizing

and drying gas flow were set 3 and 10 L/min respectively. The temperature of the interface, desolvation area, and

heart block were set at 300 °C, 250 °C, and 400 °C, respectively.

Methods for cell culturing and cytotoxicity evaluation

The cytotoxicity of CS-PEP-DOX/Cur nanoparticles on MCF-7/ADR (a DOX resistant cancer cell line) and L929

(normal cell line) was studied using a Kit-8 assay (CCK-8, Dojindo, Japan).4, 5 The cells were prepared according

to the manufacturer’s protocol. The cells were cultured in RPMI-1640 (Gibco, Grand Island, NY) supplemented

with 100 U/mL penicillin/streptomycin (Gibco) and 10% (v/v) FBS (Gibco) at 37 °C in an incubator with a

humidified atmosphere of 5% CO2 and 90% relative humidity. L929 (5×103 cells) and MCF-7/ADR (5×103 cells)

cells at their logarithmic growth phases were seeded in 96-well plate (Corning Inc., NY, USA). After 24 h

incubation, cells adhered to the bottom of the well and CS-PEP-DOX/Cur nanoparticle suspension was added

at designated concentration and incubated for another 48 h. After gentle washed with PBS, a solution of 10%

(v/v) CCK-8 in RPMI-1640 was added and incubated for 3 h. Absorbance at 450 nm was recorded by a

SpectraMax® microplate reader (M5, Molecular Devices LLC, Sunnyvale, CA). The cell survival rate was

determined by comparing treated cells absorbance to that of untreated control cells. Moreover, the cytotoxicity

of free DOX, free Cur, combined free DOX and free Cur (Free DOX/Cur), and CS-PEP-DOX nanoparticles were

evaluated using the same method at the drugs concentration corresponded to CS-PEP-DOX/Cur nanoparticles.

Data for chemical structure identification

The chemical structure of DOX-PEP(Trt)-CH2-CH2-COOH was present in Fig. S1A. As shown in Fig. S1 B and C,

has quasi-molecular ion peaks m/z [M + Na]+ 1278.1 and [M-H]- 1254.3 corresponding to a molecular weight of

1255 Da, which matched well with that of compound (3). The 1H-NMR (Fig. S1D) data for compound (3) was

present as blow: δ 0.84 ( H-32 and H-33, CH3*2), δ 1.16 (H-27, CH3), δ1.23 (H-31, H-41 and H-44, CH and CH3*2),

δ 1.82 (H-30, CH2), δ 1.95 and 1.54 (H-23, CH2), δ 1.99 (H-23 and H-46, CH2*2), δ 2.12 (H-47, CH2), δ2.29 (H-13,

CH2), δ 2.85 (H-36, CH2), δ 2.99 (H-11, CH2), δ 3.55 (H-24, H-25, H-26, CH*3), δ 3.92 (H-1, CH3), δ 4.22 (H-24 and

H-26, CH*2), δ 4.65 (H-29, H-35, H-36, H-43, CH*4), δ 4.95 (H-21, CH2), δ 5.08 (H-22, CH), δ 7.4-7.9 (Benzyl

hydrogen and H-3, H-4, H-5). These data confirmed the successful synthesis of DOX-PEP(Trt)-CH2-CH2-COOH.

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Figure S1 The chemical structure of DOX-PEP(Trt)-CH2-CH2-COOH (A), and its MS in positive model (B) and negative model (C), and 1H-NMR spectra (D).

For CS-PEP(Trt)-DOX Characterization, a KBr tablet (120 mg) containing 6 mg of CS-PEP(Trt)-DOX was

prepared, and subjected to Fourier transform–infrared (FT-IR) determination. As shown in Fig. S2, two broad

absorptions at 3446 cm-1 and 3390 cm-1 were respectively attributed to the symmetric and antisymmetric stretching

vibration of the primary amine of chitosan. The intensity peak at 1021 cm-1 belonged to C-O-Hand C-O-C

stretching vibration in sugar moiety. Peaks that appeared at 2964 cm-1 and 2850 cm-1 belonged to the C-H

stretching vibration of CH3 and CH2 groups in PEP(Trt)-DOX moiety. A typically double peak at 1385 cm-1 and

1405 cm-1 indicating a gem-dimethyl group, matched well with the characteristic leucine residue in the legumain

sensitive peptide. The peaks at 1665 cm-1 and 1550 cm-1 corresponded to the amide bond in this molecule. Peaks

at fingerprint region (803 cm-1, 730 cm-1) suggested multi-substitute aromatic ring in this molecule. These data

suggested that CS-PEP(Trt)-DOX was successful synthesized.

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Figure S2 The FT-IR spectra of CS-PEP(Trt)-DOX

The representative H&E images of the normal tissues from experimental animal with vary treatments

Figure S3 Reduced in vivo system toxicity of CS-PEP-DOX/Cur NP. The scale bar is 50 μm.

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Supplementary data for MDR overcoming effects evaluation

Figure S4 The representative images of the expression profile of MDR1 (A), MRP 1 (B), and MVP (C) in solid tumor with treatments of PBS, free DOX, or free DOX/Cur observed by CLSM. The scale bar is 20 μm.

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