Methoxy poly(ethylene glycol) amine (mPEG-NH2, Mn=5000), Polyethylene-polypropylene glycol (PLGA-mPEG, Mn=8000), doxorubicin hydrochloride (DOX) and α-tocopheryl succinate (TOS) were purchased from Macklin Company, China. N(ε)-benzyloxycarbonyl-L-lysine (Cbz-lysine) and L-methionine was purchased from Energy Chemical, China. Dimethylmaleic anhydride (DMA) was purchased from Bidepharm, China. L-lysine-N-carboxyanhydride (Cbz-Lys-NCA) were synthesized by the Fuchs-Farthing method using bis(trichloromethyl) carbonate (triphosgene) as reported earlier [6,22]. Propidium iodide and 4’, 6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma Company. Annexin V-FITC Apoptosis Detection Kit was purchased from Kengen. All other reagents and solvents were purchased from Sinopharm Chemical Reagent Co., Ltd., China.
Synthesis of methoxyl poly(ethylene glycol)-block-poly(L-lysine) (mPEG-b-PLL)
Firstly, poly(ethylene glycol)-block-poly(N(ε)-benzyloxycarbonyl-L-lysine) (mPEG-b-PLL(Z)) was synthesized by ring open polymerization (ROP) of Lys(Z)-NCA in DMF using mPEG-NH2 as initiator and the deprotection of benzyl groups as described earlier [22,23]. Typically, dried mPEG-NH2 (1.0 g, 0.2 mmol) and Cbz-Lys-NCA (1.8 g, 6.0 mmol) were added in a 50 mL dried glass reactor with 30 mL of DMF. Stirred at 25°C for 3 days, the reaction mixture was poured into 150.0 mL of ice ether for three times to precipitate pure mPEG-b-PLL(Z) polymers (Yield: 88.6%). The degree of polymerization (DP) of mPEG-b-PLL(Z) was determined to be 30 by 1H NMR spectra (Figure S1). The number-average molecular weights (Mn) of mPEG-b-PBLG polymer calculated by 1H-NMR was 12870 (mPEG5000-b-PLL(Z)7870). Subsequently, 1.6 g of mPEG-b-PLL(Z) was added into 10.0 mL of trifluoroacetic acid (TFA) and HBr/HOAc (0.8 mL) to remove Cbz groups. Stirred for 1 hour under ice bath, the mixture was precipitated into 150.0 mL of ice diethyl ether, following being dialyzed (molecular weight cut-off (MWCO) = 3500 Da) against distilled water, and then freeze-dried to obtain mPEG-b-PLL product and the yield was 75.6 %. The purified product was dried under vacuum at room temperature.
Synthesis of methoxyl poly(ethylene glycol)-block-poly(L-lysine)- graft-α-tocopheryl succinate and methionine modified with dimethylmaleic anhydride (PPT/M(DMA))
The N-(tert-Butoxycarbonyl)-L-methionine (D(Boc)) was firstly prepared as described previously . Dried mPEG-b-PLL (1.0 g, 0.11 mmol), D(Boc) (0.74 g, 3.30 mmol), NHS (1.71 g,14.85mmol) and DCC (3.06 g, 14.85 mmol) was dissolved in 30.0 mL of DMSO under nitrogen gas. Stirred at 25°C for 24 hours, the reaction solution was firstly filtered and dialyzed against DMSO and then dialyzed against distilled water, following freeze-dried to obtain methoxyl poly(ethylene glycol)-block-poly(L-lysine)-graft- methionine (Boc) (PPT/D(Boc)) product (PPD(Boc)). The number of grafted D(Boc) was 24 from 1HNMR (Figure S2). PPD(Boc) (1.15 g, 0.08 mmol), TOS (0.35 g, 0.66 mmol), N-hydroxysuccinimide (NHS, 0.15g, 1.32mmol) and N, N’-dicyclohexylcarbodiimide (DCC, 0.27 g, 1.32 mmol) was dissolved in 30.0 mL of DMSO under nitrogen gas. Stirred at 25°C for 48 hours, the reaction solution was firstly filtered dialyzed against DMSO and then dialyzed against distilled water, following being freeze-dried to obtain methoxyl poly(ethylene glycol)-block-poly(L-lysine)-graft-α-tocopheryl succinate and methionine (Boc) (PPT/D(Boc)) product (Figure S3). Subsequently, 0.8 g of PPT/D(Boc) was dissolved in 5.0 mL of DCM and 5.0 mL of TFA was added dropwise under ice bath. Stirred for 1 hour, the mixture was filtrated and precipitated into 100.0 mL of ice diethyl ether, following dialyzed against distilled water, and then freeze-dried to obtain poly(ethylene glycol)-block-poly(L-lysine)-graft-α-tocopheryl succinate and methionine (PPT/D) product. The shell was prepared by the reaction between PPT/D and DMA [25-26]. Briefly, PPT/D and double DMA were dissolved in DMSO, then triethylamine (TEA) and pyridine were added and stirring under nitrogen protection at the room temperature overnight. The mixture was purified by dialysis (MWCO 3500 Da) against DMSO for 24 hours, following being dialyzed in dialysis bag (MWCO 10,000 Da) for 24 hours to remove DMSO, and lyophilized. For comparison, succinic anhydride (SA)-modified shell (Shell-SA) was prepared in the similar route and denoted as PPT/D(SA). SA modified micelle would not undergo pH-sensitive hydrolysis and thus not offering surface charge reversal as DMA modified one does. PPD was obtained with deprotection of PPD(Boc), modified with DMA without TOS and denoted as PPD(DMA) (Figure S3). As comparison, PPT/D(SA) was synthesized and characterized as shown in Figure S4. To investigate the ROS-responsiveness, methionine was incubated with H2O2 for 0, 4 and 12 hours in D2O. The chemical changes were recorded by 1H NMR in Figure S5. Methionine and its oxidized product were observed 4 hours later. After 12-hours incubation, methionine was completely oxidized.
Micelle preparation and characterization
The solvent exchange method was used to prepare micelles in this study. DOX·HCl (5.0 mg) was dissolved in DMF (1 mL) with TEA (2.6 mg) to remove the HCl of the DOX·HCl and was stirred for 2 hours. An amount of PPT/D(DMA) was dissolved in DMF (1 mL), mixed with the above solution and then stirred in dark for another 2 hours. Then the solution was added to 5 mL PBS (pH 9.0) by using infusion pump at a constant rate of 2 mL/h and was stirred for another 3 hours. After that the solution was loaded into a MWCO 3500 dialysis bag and dialyzed against pH 8.0-9.0 water for 24 hours. The obtained solution was mildly centrifuged and the supernatant was stored in 0 °C which would be used directly. The similar process was for other micelles. The DOX loading content was determined by lyophilizing 1.0 mL of the above solution and dissolved the obtained powder in DMSO. The concentration of the DOX-loaded micelles was measured at excitation and emission wavelengths of 485 nm and 550 nm. The loading content (LC) and encapsulation efficiency (LE) of DOX micelles were calculated by the following equations.
For the characterizations of the empty micelle and DOX-loaded micelles, the particle sizes, size distributions and zeta-potentials were measured by using a Zetasizer (Malvern 3000HSA). The morphologies of the micelles were identified by transmission electron microscopy (TEM) images obtained by a JEM-2000EXII transmission electron microscope with an accelerating voltage of 200 kV. The morphology changes of micelles were also evaluated when micelles were incubated in different pH solutions or H2O2 solutions.
pH-sensitive property of PPT/D(DMA)@DOX
In order to evaluate pH-sensitive property of PPT/D(DMA), the micelle was tested compared with PPT/D(SA). Micelles (500 μg/mL) were incubated in PBS at pH 7.4 and 6.8 for 200 minutes, respectively. At predetermined intervals, the mean diameter and zeta potentials of the micelles were measured by DLS. To evaluate the pH-sensitive property of PPT/D(DMA)@DOX and PPT/D(SA)@DOX, DOX-loaded micelles were incubated at pH 7.4, 6.8 and 5.5 for 200 minutes. Average size and zeta potential were recorded by DLS.
Drug release behavior
To study the ROS responsibility of PPT/D(DMA)@DOX, 3.0 mg of micelles suspended respectively in 1.0 mL of PBS (pH=7.4) containing various concentrations of H2O2 (0, 0.1, and 10.0 mM) were sealed in a dialysis bag (MWCO 3.5 kDa). The dialysis tubes were subsequently immersed into glass tube containing 30.0 mL of PBS (pH=7.4) with same concentrations of H2O2. The released DOX and TOS were measured by a Lambda Bio40 UV/Vis spectrometer and HPLC, respectively. Likewise, the same concentration of PPT/D(DMA)@DOX and PPT/D(SA)@DOX was immersed in buffer solution with various pH values (pH 7.4, 6.8 and 5.5) at 37 °C under shaking at 100 rpm. The release media were taken out at predetermined times and an equal volume of fresh PBS was added. After that, the amounts of released DOX and TOS were detected.
Human lung adenoma cell lines A549 were purchased from the American Type Culture Collection (ATCC, Rockville). The cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin and grown in a 37°C humidified environment containing 5% CO2.
Evaluation of the ROS-responsiveness in cells
ROS level changes in cells were determined with 2’,7-dichlorofluoresceindiacetate (DCFA-DA) dye. A549 cells were seeded onto plates at a density of 1.0 × 105 cells per well. After an incubation for 24 hours, the cells were treated with empty micelles (PPT/D(DMA) or PPD(DMA)) and PPT/D(DMA)@DOX for different incubation times. And the cells without any treatment were used as control. After co-incubation for predetermined time, the cells were washed with PBS for three times and the media were replaced with DCFH-DA (10 μM) at 37 °C for 30 minutes. Finally, all the cells were observed by fluorescence microscopy and the green fluorescence intensity was measured with a microplate reader (ELIASA of Perkin Elmer) at 490 nm.
Cell apoptosis assay
To investigate the apoptotic effect of different drug formulations, A549 cells were treated with free DOX, free TOS, DOX/TOS, PPT/D(SA), PPT/D(DMA), PPT/D(SA)@DOX and PPT/D(DMA)@DOX. The concentration of DOX and TOS was set as 0.5 and 0.75 μg/mL. The extent of apoptosis in A549 was evaluated by flow cytometry (FCM) (ESP Elite, Beckman-Coulter, Miami, FL) analysis using FITC-conjugated AnnexinV/propidium iodide (PI, BD PharMingen) staining, following the manufacturer's instructions. Both early apoptotic (Annexin V-positive, PI-negative) and late apoptotic (Annexin V-positive and PI-positive) cells were included in cell death determinations.
A549 cells were seeded in the glass bottom dishes at a density of 1.0×105 cells per well for 24 hours. Then all the cells were incubated with PPT/D(SA)@DOX or PPT/M(DMA)@DOX (both containing 2.5 μg/mL DOX) for 2 and 4 hours at pH 7.4 and 6.8, respectively. Thereafter, the media were removed and the cells were washed with PBS to remove the extracellular micelles. The cell nuclei were stained by DAPI according to the standard protocol provided by the supplier. At last, the cellular uptake of samples was visualized under fluorescence microscopy. To quantitatively investigate cellular uptake, A549 cells were treated with with PPT/D(SA)@DOX or PPT/M(DMA)@DOX (both containing 2.5 μg/mL DOX) for 2 and 4 hours at pH 7.4 and 6.8, respectively. After detachment of cells, the mean fluorescence intensity of cells was detected by FCM.
Cell viability study
The cytotoxicity of free DOX, free TOS, PPT/D(SA), PPT/D(DMA), PPT/D(SA)@DOX and PPT/D(DMA)@DOX were evaluated by the MTT assay. A549 cells were seeded in 96-well plates at a density of 5000 cells per well in 100 μL DMEM containing 10 % FBS and cultured for 24 hours at 37 °C. Then the cells were treated with 100 μL culture medium containing fixed amount of micelles for 48 hours. After that the medium was replaced with 200 μL of fresh DMEM and 20 μL MTT (5 mg/mL in PBS) and incubated for another 4 hours. Then the medium was removed and 200 μL DMSO was added. The optical absorbance was measured at 450 nm of each well using a microplate reader. To detect the pH-responsive charge conversion, the cytotoxicity of PPT/D(SA)@DOX and PPT/D(DMA)@DOX was analyzed at pH 7.4 and 6.8. The cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. All the cytotoxicity tests were conducted in triplicate.
To investigate the bioditribution of blank micelles, tumor-bearing mice were intravenously injected with saline, Cy5-labelled PPT/D(SA) and PPT/D(DMA). At determined time points (12, 24 and 48 hours), mice were sacrificed and the major organs, including tumors, were collected. The fluorescent images of these tissues were taken on an infrared imaging system (Caliper, XenoFluor 750).
In vivo antitumor study and histochemistry analysis
Nude mice (5-6 weeks old) were purchased from Beijing Institution for Drug Control, China. A549 cell tumor-bearing mice model was established by subcutaneous injection of A549 cells (2×106) into the right axilla of each mouse. In vivo/ex vivo imaging and biodistribution experiments were performed at day 10 after tumor cell injection, by which time tumors had grown to 0.8 cm in diameter. At that time, A549 cell tumor-bearing nude mice were intravenously injected with saline, free DOX, PPT/D(SA)@DOX and PPT/D(DMA)@DOX micelles at a dose of 0.5 mg/kg DOX. The mice were sacrificed after 24-hours post-injection. At 24-hours post-injection, the mice were sacrificed, and the hearts, livers, spleens, lungs, kidneys, and tumors were excised to directly observe the fluorescence distribution. The emission fluorescence was collected from 450 to 700 nm, with the 455 nm excitation filter used.
Moreover, when the tumor volume reached 50 mm3, A549 tumor-bearing mice were divided into 4 groups randomly (n=4) and treated with PBS, free DOX, PPD(DMA)@DOX, TOS/PPD(DMA)@DOX, PPT/D(SA)@DOX and PPT/D(DMA)@DOX (fixed concentration of DOX at 2 mg/kg) through intravenous injection at day 0, 3, 6 and 9 respectively. For TOS/PPD(DMA)@DOX treatment, TOS at a dose of 1mg/kg  was used in combination with PPD(DMA)@DOX
Tumor volumes and body weights of all the mice were recorded every two days. Tumor volumes (V) and body weights were measured to evaluate the antitumor activity and systemic toxicity. Tumor volume (V) was calculated using the following formulas:
V= a×b2/2 (4)
Where a and b were major and minor axes of the tumors measured by a caliper, respectively and all the mice were sacrificed at day 16. Tumors were dissected, washed and used for histology analysis. Cell state in tumor tissue was analyzed by hematoxylin-eosin (H&E) staining. Simultaneously, to evaluate the levels of apoptosis in tumor areas, tumor tissues were stained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) according to the manufacturer's protocol (Roche, Penzberg, Germany). Ki-67-stained sections were observed under a microscope (X51 Olympus; Olympus Corp. Tokyo, Japan).
The results were presented as mean ± standard deviation (SD). Statistical significance was analyzed using Student’s t-test.