Drug Self-Assembled Delivery System with Dual Responsiveness for

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Drug Self-Assembled Delivery System with Dual Responsiveness for Cancer Chemotherapy Xiao Duan, Heng Chen, Li Fan, and Jie Kong ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00559 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 13, 2016

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ACS Biomaterials Science & Engineering

Drug Self-Assembled Delivery System with Dual Responsiveness for Cancer Chemotherapy Xiao Duan†, Heng Chen†, Li Fan‡, and Jie Kong†* †MOE Key Laboratory of Space Applied Physics and Chemistry, Shaanxi Key Laboratory of Macromolecular Science and Technology, School of Science, Northwestern Polytechnical University, Xi’an, 710072, P. R. China ‡Department of Pharmaceutical Chemistry and Analysis, School of Pharmacy, The Fourth Military Medical University, Xi’an, 710032, P. R. China

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ABSTRACT: In this study, we present a novel drug self-assembled delivery system (DSD) with pH and glutathione dual responsiveness to synergistically address the problems of traditional polymer-based carriers, i.e., their low drug loading efficiency, poor biocompatibility and nonbiodegradability. The DSD system with minimum assistant substances was developed from methotrexate (MTX) model drug copolymers and polyethylene glycol (PEG), which gives the system a higher drug loading efficiency and completely avoids the use of toxic carriers. The amphiphilic block copolymers of MTX and PEG are self-assembled into stable micelles such that MTX can be delivered to tumor tissues in vivo and controllable release can be achieved for cancer therapy via the cleavage of the reversible covalent bonds in the copolymer. The micelles overlapped with lysosomes for cellular uptake, and the in vivo distribution was higher in tumor tissues. Biological evaluation and histological analysis confirmed that the DSDS micelles were more effective in killing tumor cells than free MTX. In addition, there were fewer side effects in normal tissues. As a result, tumor growth could be effectively inhibited in vivo. The DSD concept is a perfect emerging strategy to address the problems of traditional polymer-based anticancer drug carriers in a synergetic manner and offers new potential routes of cancer therapy and clinical treatments.

KEYWORDS: drug delivery, disulfide bonds, imide bonds, cleavage reactions, tumor therapy


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INTROCUTION Drug delivery systems for cancer chemotherapy have been widely developed as an emerging and cross-functional field. Numerous drug delivery systems have been reported for delivering anticancer drugs to tumors based on inorganic carriers such as mesoporous silica nanocapsules,1,2 nanobubbles,3 carbon nanotubes,4,5 and organic carriers such as liposomes,6,7 dendrimers,8,9 polymer micelles or prodrugs micelles,10-12 and innovative drug-drug conjugates micelles.13 In general, the key issues of this area are controlled drug release, drug loading efficiency, and carrier biocompatibility and biodegradability in vivo. The drugs are usually encapsulated in or conjugated with various carriers. The leakage of partial encapsulated drugs unavoidably occurs during the self-assembly and dialysis of the drug delivery system. In addition, the encapsulated drugs often leak from carriers in the blood stream, especially for highly water-soluble drugs,14 resulting in low drug loading. Therefore, conjugated drugs delivery systems are considered a better method because of their stability inthe blood stream and their high drug loading. However, their controllable release is a new topic. Although the controllable release from carriers at target sites has been addressed based on pH-responsive carriers using hydrazone bonds,15,16 acetyl groups,17,18 imine bonds,19-22 poly-L-histidine,23-25 reduction-cleavable disulfide bonds,26-30 and thermo-sensitive poly(N-isopropylacrylamide),31,32 the poor biocompatible and nonbiodegradability nature of these stimuli-responsive carriers in vivo hampers the further application of encapsulated or conjugated drug delivery systems in clinical medicine.



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The components of both encapsulated and conjugated drug delivery systemsalways include polymer-based carriers.Currently, only a few polymer materials, such as polyethylene glycol (PEG) and poly(lactic acid) (PLA), have been approved by the FDA for clinical applicationsand use in humans. Other polymers, e.g., the cationic poly(amidoamine) dendrimers, are able to induce cell membrane damage resulting in the leakage of intracellular components and leading to hemolysis even at a low concentration.33 For monomethoxy(polyethylene glycol)−poly(D,L-lactic-co-glycolic acid)−poly(L-lysine) (mPEG-PLGA-PLL), the degradation property has been evaluated, and the results show that the size of mPEG-PLGA-PLL remains nearly unchanged and that the minimal molecular weight is higher than 6kDa at pH 5.0 and pH 7.4 after 144 h.34 Poly(ferulic acid-co-tyrosine) can be completely degraded within 26 days at pH 12 and 70°C.35 However, the degradation time is unacceptable for biomedical applications in both encapsulated and conjugated drug delivery systems.

However, traditional conjugated drug or prodrug delivery systems (incorporation of drugs as a driving force to assemble formulations36) are mainly based on polymer-based carriers. When designing anticancer drug delivery carriers, the use of toxic carriers and nonbiodegradability polymers should be fully avoided. On the other hand, the drug-to-polymer carrier ratio is an important consideration because the use of more polymer carriers results in poor metabolism and elimination in vivo. To solve these problems of low drug loading efficiency, synergetic drug controlled release and carrier accumulation, toxicity, biocompatibility and biodegradability, we present a novel design of a highly efficient drug self-assembled delivery system (DSD) with pH and


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glutathione (GSH) dual responsiveness in which anticancer drugs copolymerized with reduction-cleavable disulfide bonds were employed as a hydrophobic core and protected by the hydrophilic PEG linked via pH-responsive imine bonds. In this DSD, the drugs from the copolymers with drug activity can be delivered by themselves with the help of only a few PEG complexes and released in tumor cells via the cleavage of reversible covalent bonds (disulfide and imine bonds) under high concentration of GSH and low pH value. Therefore, the high drug loading efficiency, good controlled release, biocompatibility and biodegradability in comparison to polymer carriers or prodrugs micelles (including other pH/reduction dual-sensitive nano-sized drug delivery systems37,38) can be perfectly addressed in a synergetic manner, which is a potential emerging strategy in drug delivery and cancer therapy.

METHODS Materialsand instruments. N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), 1-hydroxybenzotrizole (HOBt), methoxypolyethylene glycol, and N, N′ -dicyclohexylcarbodiimide (DCC) were purchased from Aladdin Co. China. Glutathione (GSH) was purchased from Sigma Aldrich. N-[4-[[(2, 4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-l-glutamic acid (methotrexate, MTX) was purchased from Wuhan DKY Tech. Co., Ltd. (China). Cystaminedihydrochloride was purchased from TCI Co. mPEG-CHO (Mw=5,000 Da, PDI =1.04) was purchased from Shanghai Yare Biotech, Inc. Cy5-NHS was purchased from Shanghai Seebio Biotec, Inc. LysoTracker®Green DND was purchased from Shanghai QCBIO Sci. &Tech. Co., Ltd (China). N, N-dimethylformamide (DMF),



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triethylamine (TEA) and common reagents were purchased from Guangzhou Jinhuada Chemical Reagent Co., Ltd. and used without further purification. The micelle size was determined by dynamic light scattering (DLS) using a Malvern Zetasizer ZS90 instrument (Worcestershire, U.K.). TEM images of all micelles were obtained through a FEI Tecnai G2 F20 S-Twin TEM (Hillsboro, OR). Cellular uptake was characterized with a Zeiss LSM510 confocal laser microscope (Carl Zeiss Shanghai Co. Ltd, Shanghai, China) and an Attune® acoustic focusing cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA). The molecular weight was measured by Gel Permeation Chromatograph waters 1515, and the standard reagent was polymethylmethacrylate (PMMA). Measurements of NMR spectra were conducted using a Bruker Avance 400 spectrometer (Bruker BioSpin, Germany) operating at 400 MHz (1H) in DMSO-d6.The flow cytometry experiment and animal experiment (including measurement machines and materials) were conducted by Redland bio-company in Guangzhou of China.

Preparation of MTX-S-S drug copolymer. Methotrexate (500 mg, 1.1 mmol), EDC·HCl (2.2 mmol) and (Hobt, 2.2 mmol) were dissolved in DMF (20 mL) in a 100 mL round bottom flask. Then, the cystaminedihydrochloride (248 mg, 1.102 mmol) was put into a flask one hour later. Then, the reaction was carried out at room temperature for 24 h. TEA was added to the flask to react for another further 24 h. DMF was concentrated to 5 mL, and the product was precipitated three times in THF. The yellow precipitation was collected and dried as the MTX-S-S drug copolymer (yield: 80%). NMR (400 MHz, DMSO-d6) spectra were taken with trimethylsilane (TMS) as an internal reference standard. 1H-NMR ppm: 8.55 (s, 1H), 8.09 (d, 1H), 7.72 (d, 2H), 6.79 (d, 2H), 4.77 (s, 2H), 4.34 (m,


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1H), 3.28 (t, 4H),3.12 (s, 3H), 2.74 (t, 4H), 2.16 (t, 2H), 1.88–2.00 (m, 2H).13C-NMR ppm: 28.07, 32.59, 37.58, 38.46, 39.7, 53.84, 55.38, 111.54, 121.84, 122.40, 149.34, 149.84, 151.20, 158.95, 163.14, 166.53, 172.46, 174.34.13C-1H COSY spectra are shown in Figures S1-S2.

Preparation of MTX-hexamethylenediamine drug copolymer. Methotrexate (500 mg, 1.1 mmol), EDC·HCl (2.2 mmol) and (Hobt, 2.2 mmol) were dissolved in DMF (20 mL) in a 100 mL round bottom flask. Then, hexamethylenediamine (128 mg, 1.102 mmol) was added to the flask one hour later. The reaction was carried out at room temperature for 24 h. TEA was added to flask to react for another 24 h. DMF was concentrated to 5 mL, and the product was precipitated three times in THF. The yellow precipitation was collected and dried as the MTX-hexamethylenediamine drug copolymer (yield: 60%). NMR (400 MHz, DMSO-d6) spectra were taken with trimethylsilane (TMS) as an internal reference standard. 1H-NMR ppm: 8.59 (s, 1H), 8.00-8.11 (d, 2H), 7.72 (d, 2H), 6.97 (d, 2H), 4.80 (s, 2H), 4.33 (m, 1H), 3.21 (s, 3H), 2.90 (t, 4H), 2.14 (t, 2H), 1.88–2.00 (m, 2H),1.33 (m, 4H),1.17 (m, 4H). 13C-NMR ppm: 26.56, 28.25, 29.47, 31.25(DMF), 32.65, 36.27(DMF), 38.90, 39.63, 53.85, 55.33, 56.51, 111.50, 121.79, 122.06, 129.42, 147.50, 149.52, 151.27, 153.84, 162.04, 162.80 (DMF), 163.18, 166.49, 172.11. 13C-1H COSY spectra are shown in Figures S3-S5.

Preparation of amphiphilic block copolymer of MTX-S-S-PEG-CHO from MTX-S-S and mPEG-CHO. MTX-S-S (200 mg) and mPEG-CHO (200 mg) were dissolved in DMF (10 mL) in a 25 mL round bottom flask, and then a few drops of acetic acid were dropped into the flask with molecular sieves. The reaction was carried out at room temperature for 24 h. The product was



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precipitated in THF. The amphiphilic block MTX-S-S-PEG product was dissolved in water to form micelles. The micelles were a freeze-dried powder (yield: 25%). 1H-NMR ppm: 3.51 (4H,-CH2CH2O-).13C-NMR ppm: 70.25 (-CH2CH2O-) (other position refers to the spectra of MTX-S-S).13C-1H COSY spectra are shown in Figures S6-S7.

Preparation of amphiphilic block copolymer of MTX-hexamethylenediamine-PEG-OH from MTX-hexamethylenediamine and mPEG-OH. MTX-hexamethylenediamine (200 mg),mPEG-OH (200 mg), DCC (124 mg), and DMAP (44 mg) were dissolved in DMF (10 mL) in a 25 mL round bottom flask.Then, the reaction was carried out at room temperature for 72 h. DMF was concentrated to 5 mL, and the product was precipitated three times in THF. The yellow precipitation was collected and dissolved in pure water.The micelleswere extracted by centrifugingat 12000rpm, and then, unreacted MTX-hexamethylenediamine was collected at the bottom of the centrifuge tube. The supernatant was freeze-dried and the ultimate yellowish product was obtained (yield: 10%). The NMR spectrum is shown in Figure S8.

Preparation of the fluorescently labeled MTX-S-S-PEG-CHO by Cy5. Cy5-NHS (1 mg) and MTX-S-S-PEG (60 mg) were dissolved in DMF (2 mL) and reacted for 24 h at room temperature. Then, the product was precipitated three times in THF. The green power was collected.

Measurement of critical micelle concentration of MTX-S-S-PEG-CHO. The critical micelle concentration (CMC) was determined by a fluorescence technique (an excitation wavelength was


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335 nm). A calculated volume of the pyrene solution in acetone was added to a series of volumetric flasks, acetone was removed under reduced pressure, polymer solutions at different concentrations were then added to the volumetric flasks, and the pyrene concentration was fixed at 6×10-6 mol·L-1. All the samples were allowed to stand for 1 day before fluorescence measurement. The measured CMC was 22 µg·mL-1 (Figure S9).

Cumulative release of MTX. MTX-S-S-PEG-CHO and MTX-hexamethylenediamine-PEG-OH were dissolved in 2 mL phosphate-buffered saline (PBS) (pH=7.4) in a dialysis bag (MW=1,000 Da) and then put into flasks with 28 mL PBS under different conditions (pH=7.4, pH=7.4 and GSH=1 mg·mL-1; pH=6.0 and GSH=1.0 mg·mL-1). Three milliliters PBS was pipetted at different time intervals in each flask, and then 3 mL fresh PBS was added. The absorbance of MTX was determined using a UV-vis spectrometer (Figure S10).

Cell apoptosis analysis. Cervical cancercells (Hela) and human breast adenocarcinoma cell line (MCF-7) cells were seeded at 1×105cells per well in 6-well plates and incubated for 24 h before treatment with MTX and MTX-S-S-PEG (equivalent doses applied) for another 24 h and stained using an Annexin V-FITC Apoptosis Detection Kit (Cat No. 556547, BD, USA) consisting of FITC-conjugated Annexin V and propidium iodide (PI), following the manufacturer's instructions. Cell apoptosis was analyzed by flow cytometry (BD FACS).



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Cellular uptake assay. Iaryngeal carcinoma Hep2 cells were seeded with a density of 5×104 per dish in 35 mm glass microscopy dishes and incubated overnight at 37°C. Then, the Hep2 cells were treated with 4 mg·mL-1 Cy5 fluorescently labeled MTX-S-S-PEG micelles at pH = 7.4 to confirm the distribution of the micelles. Then, the cells were stained with Lyso Tracker® Green DND for 4 h, gently rinsed with PBS three times and observed via confocal laser scanning microscopy (CLSM).

Animal experiment. Ishikawa tumor-bearing mice were divided into 3 groups, minimizing the differences in weights and tumor sizes in each group. Animal experiments were conducted in accordance with local Institutional Animal Care and Use Committee (IACUC) guidelines. To evaluate the toxicity profile of free MTX and Cy5-MTX-S-S-PEG-CHO in vivo, we used approximately 6- to 8-week-old Ishikawa nude mice. The mice were inoculated subcutaneously in their left flank with 5 ×106Ishikawa cells dispersed in PBS and injected with MTX (2.5 mg·kg-1) and Cy5-MTX-S-S-PEG-CHO (equivalent dose). The Ishikawa tumor-bearing nude mice were intravenously administered with Cy5-labeled MTX-S-S-PEG and free MTX at 1 day, 4 days, 8 days and 12 days. One mouse in the saline and MTX group died on the fourteenth day, and the tumor volume was measured on the fourteenth day.

In vivo imaging. To trace the distribution of DSDS in the tumor-bearing mice, the mice were intravenously administered with Cy5-MTX-S-S-PEG-CHO, and fluorescence images were observed 6 h after administration. For histology, 6 h after administration, the mice were sacrificed, and the harvested tumors and main organs were frozen in OCT, cut into 10 mm slices, and stained with


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4',6-diamidino-2-phenylindole (DAPI) (5 mg·mL-1) for 5 min. Tumor and organ slices were fixed in 4% paraformaldehyde for 20 min and sealed with a coverslip. The samples were analyzed using CLSM.

Histological analysis. For histology, the tumor and main organs were removed from the drug-treated mice at 16 days, fixed in 10% paraformaldehyde and embedded in paraffin blocks. To analyze cell apoptosis, a TUNEL assay was performed using a Dead End TM Colorimetric Apoptosis Detection System (Promega, Madison, WI) according to the manufacturer's instructions. Apoptotic cells were visualized under an Olympus microscope equipped with a computer-controlled digital camera (magnification, 250×, Tokyo, Japan).

RESULTS AND DISCUSSION Construction strategy of DSD. In detail, DSD was integrated from the drug copolymers (MTX-S-S) of methotrexate (MTX) and bis-(2-aminoethyl)disulfide and PEG linked via pH-responsive imine bonds. Therefore, the amphiphilic block copolymer (MTX-S-S-PEG) of MTX-S-S and PEG could be self-assembled into stable micelles with suitable nano dimensions for drug self-delivery and cancer therapy. The synthesis route of MTX-S-S-PEG is shown in Scheme 1. The carboxyl groups of the model drug MTX reacted with the amine groups of bis-(2-aminoethyl)disulfidedihydrochloride, and MTX-S-S was synthesized via a condensation reaction. The amine groups of MTX, i.e., drug activity sites, did not react with carboxyl groups when bis-(2-aminoethyl)disulfide was present. Then, PEG



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was linked to MTX-S-S by the formation of imine bonds between its aldehyde and the terminal amine group of MTX-S-S, as illustrated in Scheme 1.

The weight-average molecular weights (Table S1 and Figures S11-S12, PMMA standard) of MTX-S-S (Mw=7,200 Da, PDI=1.42) and MTX-S-S-PEG (Mw=10,800 Da, PDI=1.79) illustratedthat no more than one chain of PEG(Mw=5,000 Da, PDI =1.04) was conjugated because the amine groups of MTX possessed low activity to react with the aldehyde groups of PEG in comparison with bis-(2-aminoethyl)disulfide. Dynamic light scattering (DLS) of the MTX-S-S-PEG micelles self-assembled in phosphate buffer solution showed that the distribution was uniform and consistent with the TEM results (Figure 1). The polydispersity of the micelles was 0.09, and the Z-average size was 117.3 nm. The MTX-S-S-PEG micelles greatly improved the solubility of MTX and are stable in water upon centrifugation at 12,000 rpm (>50-foldt that of free MTX), which ensures therapeutic efficacy after administering a sufficient dose of the drug.

Before performing biology experiments, the loading efficiency of MTX was determined by NMR and GPC. Because no more than one chain of PEG is conjugated with MTX-S-S, the 114 repeat units of PEG (-CH2CH2O-) possess 113×4 protons. According to the NMR result, the e position has 2 protons, and the k position has 72 protons based on the position of e (Figure 2). Therefore, there are 6 repeat units of MTX. The theoretical molecular weight of the hydrophobic block is 3,500 Da. Thus, the MTX loading efficiency is at least 32%. The drug loading efficiency of traditional drug delivery systems is very low because the carriers are a significant component of drug delivery systems. Our


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DSS design avoids the application of more polymers and other unnecessary components in the carriers and greatly promotes the drug loading efficiency.

Biological evaluation in vitro. To evaluate drug release in tumor cells, a series of drug release studies in vitro were conducted. To further confirm the controlled drug released properties, we prepared additional MTX-hexamethylenediamine-PEG micelles as acontrol group. The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively.36 The concentration of glutathione inside tumor cells (approximately 2–10 mM) was higher than in extra cellular fluids and in the circulation (approximately 2–20 µM),37 while the pH was lower in the tumor cells than in the blood stream. Cleavage of the disulfide bonds and imine bonds is triggered by GSH and pH in tumor cells, respectively. After 72 h, the cumulative drug release rate of MTX-S-S-PEG was increased to 89% under the conditions of pH=6.0 and GSH=1 mg mL-1 in PBS. The cumulative drug release rate only achieved 33% under the condition of pH=7.4 PBS buffer (Figure 3).

The cumulative drug release rates of the control MTX-hexamethylenediamine-PEG group were not significantly different under the conditions of pH=7.4/GSH=1 mg·mL-1 or pH=6.0/GSH=1.0 mg·mL-1 in PBS compared with MTX-S-S-PEG-CHO in pH=7.4 PBS buffer. The size of the MTX-hexamethylenediamine-PEG micelles was not significantly different at a high concentration of GSH and a low pH value after 72 h. Conversely, the size of the MTX-S-S-PEG-CHO micelles as detected by DLS was dispeared at high GHS concentration and low pH after 72 h (Figure S13).



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These results confirmed that the disulfide bonds and imine bonds were disrupted under the conditions of high GSH concentration and low pH. The MTX drug release profiles of the micelles displayed a pH-sensitive and GSH reduction dual responsiveness, and the increased release rate was responsive to decreased pH and increased GSH concentration.

Next, we evaluated the toxicity of the MTX-S-S-PEG-CHO micelles to MCF-7 and Hela tumor cells in comparison with free MTX. Cells apoptosis assessed by flow cytometry showed that the MCF-7 cells are more sensitive to MTX. The MCF-7 cell survival rate was 76.5% upon treatment with free MTX (12 µg·mL-1), and the MCF-7 cell survival rate was 75.6% upon treatment with MTX-S-S-PEG-CHO (equivalent dose to MTX) (Figure 4A, Figures S13-14). The Hela cell survival rate was85.1% upon treatment with free MTX (18 µg·mL-1), and the Hela cell survival rate was 85.5% upon treatment with MTX-S-S-PEG-CHO (an equivalent dose of MTX) (Figure 4B). The MTX-S-S-PEG-CHO micelles possess the same drug efficacy to MCF-7 and Hela cells as free MTX. These results confirm that the drug activity sites (amine groups) of MTX are not influenced by the reaction between carboxyl groups and amine groups of bis-(2-aminoethyl)disulfide. The distribution of micelles in tumor cells was observed by confocal laser scanning microscope (CLSM) (Figure 4C). The tumor cells were stained by Lyso Tracker Green DND. The green region represents lysosomes, and the red region represents the Cy5 fluorescently labeled MTX-S-S-PEG-CHO micelles. The merge dimage illustrates that almost all micelles were located in lysosomes by endocytosis.


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Biological evaluation in vivo. In blood circulation, micelles are generally rapidly phagocytized by macrophages or captured by the complement system as exogenous substances and cleared by the kidney. The PEG hydrophilic section provided protection of the hydrophobic core and prolonged the circulation time in the blood. The “stealth” effect allowed more micelles to accumulate in tumor tissue via the enhanced permeation and retention (EPR) effect. The distributions of Cy5-MTX-S-S-PEG-CHO in vivo were investigated by CLSM 6 h after intravenous administration via the tail vein. The red sections represent Cy5-MTX-S-S-PEG-CHO. The nucleuses were stained with 4', 6-diamidino-2-phenylindole (DAPI). There was slight red fluorescence in the liver, kidney, lung, and spleen. The red fluorescence was much more widely distributed in heart tissue. The distribution of red fluorescence was widest in the tumor tissue (Figure 5A), which corresponds to the results of the TUNEL assay (Figure 6). The Cy5-MTX-S-S-PEG-CHO micelles circulated in the blood stream and accumulated in tumor tissue via EPR. The critical micelle concentration was 22 µg·mL-1, as shown in Figure S9, which ensured that the assembled micelles circulated in the blood.

To confirm authentic tumor inhibition in vivo, we selected the tumor-bearing mice with initial tumor volumes of 300-350 mm3, which are much larger tumors than reported in other studies38-41 because most of patients in the clinic were diagnosed with malignant tumors in the advanced or terminal stage. The larger initial tumor volume means that the tumor tissues are more difficult to inhibit, and the results will be more valuable in future clinical medicine. The treatment efficacies of MTX-S-S-PEG and MTX after administration in vivo (free MTX 2.5 mg·kg-1) at 1, 4, 8, and 12 days are presented in Figure 5B. The tumor volume of the control group was four-fold increased after 16



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days compared with 0 day, whereas the tumor volume of the MTX-S-S-PEG group was two-fold increased after 16 days compared with 0 day (Figure 5B). The tumor volume of the group MTX-S-S-PEG slightly increased, likely because the larger tumor is only minimally inhibited, and the DSS is administered at longer intervals between dosing. A tumor volume difference between the MTX and MTX-S-S-PEG groups was noticed at 16 days. Therefore, the MTX-S-S-PEG micelles can effectively inhibit tumor growth, resulting in a decrease in tumor weight to 34% in comparison with the control group (Figure 5C).

Histological analysis.To further confirm the treatment efficacy and side effects in vivo, a TUNEL assay was conducted. The mice were sacrificed 16 days later, and the major organs were harvested after treatment for physiopathology studies. The TUNEL results demonstrated no noticeable histological change in all tissues for the MTX-S-S-PEG-CHO group, indicating no or only slight organ toxicity of the DSD systems. The side effects of free MTX to the heart, liver, spleen, lung and kidney were obvious compared with the control group and the MTX-S-S-PEG-CHO group, especially in the spleen, lung and kidney. We can clearly distinguish dead cells from normal cells in the free MTX group (Figure 6). The MTX-S-S-PEG-CHO micelles more effectively killed tumor cells and possess a low toxicity to normal tissue compared with free MTX, likely because the micelles can accumulate in tumor tissue via the EPR effect. These results corresponded to the tumor growth inhibition and the distribution of Cy5-MTX-S-S-PEG-CHOin vivo described above. Thus, the biological evaluation in vivo, including authentic tumor inhibition and the histological analysis,


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demonstrate that the MTX-S-S-PEG-CHO micelles have fewer side effects in normal tissues and inhibit of tumor tissues in vivo compared with free MTX.

CONCLUSION In summary, we successfully developed novel drug self-assembled delivery systems with pH and GSH dual responsiveness that possess a high drug loading efficiency, good controlled release, biocompatibility and biodegradability. The drug accumulation release of the model drug MTX-based DSD was effectively released in a controllable manner, and the MTX copolymer in DSDS showed almost the same drug efficacy as free MTX in vitro. The DSDS micelles overlapped with lysosomes for cellular uptake, and the in vivo distribution was higher in tumor tissues. The histological analysis confirmed that the micelles more effectively killed tumor cells than free MTX and induced less side effects than normal tissues. Furthermore, tumor growth was effectively inhibited in vivo. The DSD concept is expected to be general and available for various anticancer drugs and addresses the problems of traditional polymer-based drug delivery carriers in a synergetic manner. These micelles possess potential applications in clinical medicine as an effective cancer chemotherapy strategy.

ASSOCIATED CONTENT

Supporting Information.

This information is available free of charge via the Internet at http://pubs.acs.org/. Details on the 1H NMR, 13C NMR, 13C-1H COSY, GPC, critical micelle concentration, UV-vis of MTX and



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MTX-S-S-PEG-CHO, Size distributions of MTX-S-S-PEG-CHO and MTX-hexamethylenediamine-PEG micelles after 72 hours of cumulative drug release, Flow cytometric analysis of cell apoptosis in MCF-7 cells and Hela cells.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected], Tel. (fax): +86-29-88431621

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support of this research by the National Natural Science Foundation of China (21374089/21404085).

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Figures Captions

Scheme 1The synthetic route of MTX-S-S-PEG and self-assembled micelles were delivered to tumor tissues via the Enhanced Eermeation and Retention (EPR) effect after being administered tail vein injection.

Figure 1 TEM images obtained by drying the aqueous dispersion of micelles (left) and the size distribution of MTX-S-S-PEG micelles measured by DLS (right).

Figure 21H NMR spectra of methotrexates (bottom) and their copolymers of MTX-S-S (middle) and MTX-S-S-PEG (top).

Figure 3 The cumulative release rate of MTX from the MTX-S-S-PEG-CHO and MTX-Hexamethylenediamine-PEG micelles in 10mM PBS buffer at 37°C. The selected concentration of GSH based on the literature.40 The increased release rate of MTX from the MTX-S-S-PEG-CHOmicelles was typically responsive to the decreased pH and increased GSH concentrations.

Figure 4 Flow cytometric analyses of cell apoptosis in MCF-7 (A) and Hela (B) cells treated with MTX and MTX-S-S-PEG micelles (equivalent dose) at 24 h, CLSM images of laryngeal carcinoma Hep2 (see Supporting Information, magnification, 400×) cells incubated with Cy5-labeled MTX-S-S-PEG micelles at 37°C for 4 h (C).



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Figure 5 The distribution of Cy5-MTX-S-S-PEG micelles 6 h after administration in Ishikawa tumor-bearing mice in heart, liver, spleen, lung, kidney and tumor tissue (A), tumor growth inhibition curves of mice treated with saline, MTX and MTX-S-S-PEG micelles after 16 days (n=5) (B), and the average weight of tumor tissue after 16 days (n=5) (C).

Figure 6 The results of the TUNEL assay in mice treated with saline, MTX and MTX-S-S-PEG micelles (magnification, 250×, red circles indicate dead cells).


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Scheme 1The synthetic route of MTX-S-S-PEG and self-assembled micelles were delivered to tumor tissues via the Enhanced permeation and Retention (EPR) effect after being administered tail vein injection.



20

15

100 Size(nm)

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Intensity (%)

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Figure 1 TEM image obtained by drying the aqueous dispersion of micelles (left) and Size distribution of micelle MTX-S-S-PEG measured by DLS (right).


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q S

H 2N

S

H N

w

H N

R O

O

S

S

O

N

n

k

m

O

k

DMSO

e

w

g NH 2

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h

9

c

OH

b N H

e

N

HN

O

q

d 8

7

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a 4

3

2

1

Chemical Shift (ppm)

Figure 2 1H NMR spectra of methotrexates (bottom) and their copolymers of MTX-S-S (middle) and MTX-S-S-PEG (top).



100% MTX-S-S-PEG-CHO pH=7.4

Cumulative release rate of MTX

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80%

MTX-S-S-PEG-CHO pH=7.4 GSH=1mg ml

-1

MTX-S-S-PEG-CHO pH=6.0 GSH=1mg ml

-1

-1

MTX-Hexamethylenediamine-PEG pH=7.4 GSH=1mg ml

-1

MTX-Hexamethylenediamine-PEG pH=6.0 GSH=1mg ml

60%

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0% 0

10

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30

40

50

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Time (h)

Figure 3 The cumulative release rate of MTX from the MTX-S-S-PEG-CHO and MTX-Hexamethylenediamine-PEG micelles in 10mM PBS buffer at 37°C. The selected concentration of GSH based on the literature.40 The increased release rate of MTX from the MTX-S-S-PEG-CHO micelles was typically responsive to the decreased pH and increased GSH concentrations.


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Figure 4 Flow cytometric analyses of cell apoptosis in MCF-7 (A) and Hela (B) cells treated with MTX and MTX-S-S-PEG micelles (equivalent dose) at 24 h, CLSM images of laryngeal carcinoma Hep2 (see Supporting Information, magnification, 400×) cells incubated with Cy5-labeled MTX-S-S-PEG micelles at 37°C for 4 h (C).



1.2

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B

saline free MTX MTX-S-S-PEG

C saline free MTX MTX-S-S-PEG

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1000 800 600 400

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Figure 5 The distribution of Cy5-MTX-S-S-PEG micelles 6 h after administration in Ishikawa tumor-bearing mice in heart, liver, spleen, lung, kidney and tumor tissue (A), tumor growth inhibition curves of mice treated with saline, MTX and MTX-S-S-PEG micelles after 16 days (n=5) (B), and the average weight of tumor tissue after 16 days (n=5) (C).


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Figure 6 The results of the TUNEL assay in mice treated with saline, MTX and MTX-S-S-PEG micelles (magnification, 250×, red circles indicate dead cells).



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For Table of Contents Use Only

Drug Self-Assembled delivery System with Dual Responsiveness for Cancer Chemotherapy Xiao Duan, Heng Chen, Li Fan, and Jie Kong*

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Drug Self-Assembled Delivery System with Dual Responsiveness for

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