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Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy
Acta Biomaterialia xxx (2016) xxx–xxx
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Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy Zhen-Gang Ma a, Rui Ma a, Xiao-Lin Xiao b,c, Yong-Hui Zhang b,c, Xin-Zi Zhang b,c, Nan Hu b,c, Jin-Lai Gao b,c, Yu-Feng Zheng a, De-Li Dong b,c, Zhi-Jie Sun a,⇑ a
Institute of Materials Processing and Intelligent Manufacturing & Center for Biomedical Materials and Engineering, Harbin Engineering University, PR China Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, PR China c Translational Medicine Research and Cooperation Center of Northern China, Heilongjiang Academy of Medical Sciences, Harbin Medical University, PR China b
a r t i c l e
i n f o
Article history: Received 26 April 2016 Received in revised form 10 August 2016 Accepted 16 August 2016 Available online xxxx Keywords: Colon-targeted Dimethyl fumarate Micelles Olsalazine Colon cancer cells
a b s t r a c t Colon-targeted drug delivery and circumventing drug resistance are extremely important for colon cancer chemotherapy. Our previous work found that dimethyl fumarate (DMF), the approved drug by the FDA for the treatment of multiple sclerosis, exhibited anti-tumor activity on colon cancer cells. Based on the pharmacological properties of DMF and azo bond in olsalazine chemical structure, we designed azo polymeric micelles for colon-targeted dimethyl fumarate delivery for colon cancer therapy. We synthesized the star-shape amphiphilic polymer with azo bond and fabricated the DMF-loaded azo polymeric micelles. The four-arm polymer star-PCL-azo-mPEG (sPCEG-azo) (constituted by star-shape PCL (polycaprolactone) and mPEG (methoxypolyethylene glycols)-olsalazine) showed self-assembly ability. The average diameter and polydispersity index of the DMF-loaded sPCEG-azo polymeric micelles were 153.6 nm and 0.195, respectively. In vitro drug release study showed that the cumulative release of DMF from the DMF-loaded sPCEG-azo polymeric micelles was no more than 20% in rat gastric fluid within 10 h, whereas in the rat colonic fluids, the cumulative release of DMF reached 60% in the initial 2 h and 100% within 10 h, indicating that the DMF-loaded sPCEG-azo polymeric micelles had excellent colon-targeted property. The DMF-loaded sPCEG-azo polymeric micelles had no significant cytotoxicity on colon cancer cells in phosphate buffered solution (PBS) and rat gastric fluid. In rat colonic fluid, the micelles showed significant cytotoxic effect on colon cancer cells. The blank sPCEG-azo polymeric micelles (without DMF) showed no cytotoxic effect on colon cancer cells in rat colonic fluids. In conclusion, the DMF-loaded sPCEG-azo polymeric micelles show colon-targeted DMF release and anti-tumor activity, providing a novel approach potential for colon cancer therapy. Statement of Significance Colon-targeted drug delivery and circumventing drug resistance are extremely important for colon cancer chemotherapy. Our previous work found that dimethyl fumarate (DMF), the approved drug by the FDA for the treatment of multiple sclerosis, exhibited anti-tumor activities on colon cancer cells (Br J Pharmacol. 2015 172(15):3929-43.). Based on the pharmacological properties of DMF and azo bond in olsalazine chemical structure, we designed azo polymeric micelles for colon-targeted dimethyl fumarate delivery for colon cancer therapy. We found that the DMF-loaded sPCEG-azo polymeric micelles showed colon-targeted DMF release and anti-tumor activities, providing a novel approach potential for colon cancer therapy. Ó 2016 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
Abbreviations: DMF, dimethyl fumarate; OLZ, olsalazine; PCL, polycaprolactone; mPEG, methoxypolyethylene glycols; EDC, 1-(3-dimethylaminopropyl)-3-ethylcar bodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; sPCEG, star-PCLmPEG; sPCEG-azo, star-PCL-azo-mPEG; TEM, transmission electron microscopy. ⇑ Corresponding author at: Institute of Materials Processing and Intelligent Manufacturing, Center for Biomedical Materials and Engineering, Harbin Engineering University, 145 Nantong Street, Nangang District, Harbin 150001, PR China. E-mail address: [email protected] (Z.-J. Sun).
1. Introduction Colon cancer is one of the most prevalent diseases, and remains a significant cause of morbidity and mortality worldwide. Despite advances in chemotherapy, resistance to the anti-cancer drugs is still the greatest challenge in the management of colon cancer.
http://dx.doi.org/10.1016/j.actbio.2016.08.021 1742-7061/Ó 2016 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
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The main drugs for chemotherapy of colon cancer include 5-fluorouracil (5-FU), capecitabine, tegafur, irinotecan, and oxaliplatin etc. However, resistance to these chemotherapies has arisen and the molecular mechanisms include decreased intracellular drug concentration, altered metabolism, or alterations of targets for the therapy [1]. Recent studies suggest that inducing necroptosis of colon cancer cells and colon-targeted drug delivery could increase the efficacy of anticancer drugs and decrease the drug resistance [2–4]. Necroptosis is a type of cell death different with apoptosis [5]. Necroptosis is characterized with (1) a morphology of necrotic cell death; (2) loss of plasma membrane integrity; (3) loss of mitochondrial membrane potentials; (4) elevation of reactive oxygen species; and (5) that the cell death was prevented by a small molecule, necrostatin-1 [5]. Han et al. proved that inducing necroptosis had a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance [5]. Grassilli et al. reported that, in drug-resistant colon carcinoma cells, induction of necroptosis by GSK3B silencing resensitized drug-resistant cells to chemotherapy [6]. Therefore, induction of necroptosis would be an approach to circumvent cancer drug resistance. Dimethyl fumarate (DMF) is the methyl ester of fumaric acid and has been approved by the FDA and European Medicines Agency as a new oral drug for the treatment of multiple sclerosis [7,8]. DMF also exhibits anti-tumor effects, for instance, it inhibits melanoma growth and metastasis [9] and induces apoptosis in HT29 colon carcinoma cells [10]. Our previous work found that DMF but not its metabolite MMF induced necroptosis in colon cancer cells through a mechanism involving the depletion of GSH, increase of ROS and activation of MAPKs [11], indicating that DMF would be a necroptosis inducer which circumvent colon cancer drug resistance. However, it should be noted that DMF could be easily hydrolyzed to mono-methylfumarate (MMF) which showed no cytotoxic effect on colon cancer cells [11]. Therefore, it must be avoided that DMF was hydrolyzed in stomach and small intestine if DMF was attempted to be developed as a local chemotherapeutic drug for colon cancer. Presently, several strategies for colon-specific drug delivery have been explored, including the techniques dependent on pH, time, pressure and/or bacteria [12]. Colonic microfloras consist of anaerobic bacteria that are only present in the colon region and secrete specific biodegradable enzymes, this feature can be exploited to target colonic drug release. Olsalazine (OLZ) is a dimer of 5-aminosalicylic acid that are linked via an azo bond. When OLZ reaches the colon part, it is cleaved by azoreductase specifically secreted by colonic bacteria [12]. Azo bond in OLZ structure indicates that OLZ can be used as a bacteria-triggered, colon-targeted materials. Therefore, in the present study, we design DMF-loaded OLZ-linked polymer micelles (Fig. 1A) and investigate the DMF release and anti-tumor activity of these micelles. Our work will provide a novel approach for colon cancer therapy.
product olsalazine was dried with vacuum at 60 °C. All other chemicals were purchased from commercial supplier.
2. Materials and methods
2.3. Assembly of polymeric micelles
2.1. Materials
The blank micelles of sPCEG-azo and sPCEG polymers were fabricated by the evaporation method. In brief, the polymer solution containing 20 mg sPCEG-azo or sPCEG in 20 mL tetrahydrofuran (THF) was added dropwisely into 40 mL deionized water with the rotor stirring. The micelle solution was filtered by 0.45 lm organic filter membrane after evaporating off THF at room temperature. The method to fabricate dimethyl fumarate (DMF)-loaded micelles was same as that for the blank micelles. The anticancer
e-Caprolactone (e-Cl), erythritol, and all the catalysts were purchased from Aladdin@ company (Shanghai, China). mPEG (Methoxypolyethylene glycols) (Mn = 2 kDa) was purchased from Energy Chemical. Co. Ltd (Shanghai, China), Olsalazine Sodium (OLZ-Na) was purchased from Jusheng Technology. Co. Ltd (Hubei, China). OLZ-Na was acidized with hydrochloric acid and the
2.2. Synthesis procedures 2.2.1. Synthesis of the star-shape PCL (polycaprolactone) The star-shape PCL was synthesized by ring-opening polymerization as described in previous work with modification [13]. e-Cl was purified by reduced pressure distillation after dried with 4A molecular sieve and added into a 100 mL round-bottom flask followed by adding Sn(oct)2 as catalyst and erythritol as initiator. The flask together with its content was degassed under vacuum for 5 h at room temperature, then the ring-opening polymerization reaction was processed at 140 °C for 6 h. After the reaction system cooled down, the products were dissolved in dichloromethane (DCM) and precipitated with cold ethyl alcohol before filtration. Finally, the star-shape PCL polymers were obtained after vacuum drying for 24 h. 2.2.2. Synthesis of methoxypolyethylene-olsalazine (mPEG-OLZ) The polymerization procedures were as following: mPEG and excess olsalazine (OLZ) were dissolved in N,N-dimethylformamide followed by adding 1-(3-dimethylaminopropyl)-3-ethylcarbodii mide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) as catalysts into the reaction system, the reaction processed at 35 °C for 48 h. Then, the product was precipitated with cold diethyl ether before filtration and redissolved in N, N-dimethylformamide, the solution was transferred into a dialysis tube (MWCO = 1 kDa) and immersed in deionized water to remove the excess catalysts and the un-reacted OLZ. Finally, the mPEG-OLZ polymers were obtained after lyophilized. 2.2.3. Synthesis of mPEG-COOH The mPEG-COOH was synthesized as described in previous work [14]. In brief, mPEG, butanedioic anhydride and the catalyst DMAP were added in a two-neck round-bottom flask and dissolved in dichloromethane, then the reaction processed at room temperature for 24 h. Next, the reactant solution was precipitated with cold diethyl ether before filtration. Finally, the products were obtained after vacuum drying for 24 h. 2.2.4. Synthesis of star-PCL-azo-mPEG (sPCEG-azo) and star-PCLmPEG (sPCEG) To synthesize the polymers with azo linkage or without azo linkage, the star-shape PCL and excess mPEG-OLZ or mPEGCOOH were dissolved in N,N-dimethylformamide followed by adding EDC and DMAP. The reaction processed at 35 °C for 48 h. Then, the reactant solution was precipitated with cold diethyl ether before filtration, and the product was redissolved in N,N-dimethylformamide. The solution was transferred into dialysis tube (MWCO = 3.5 kDa) to remove the unreacted reactants, catalysts, and N,N-dimethylformamide. Finally, the star-PCL-azomPEG (sPCEG-azo) polymers and star-PCL-mPEG (sPCEG) polymers were obtained after lyophilized. The total synthesis routes of the star-shape amphiphilic polymers were shown in Fig. 1B.
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Fig. 1. (A) The design schematic diagram of the project. DMF, dimethyl fumarate. (B) The total synthesis routes of sPCEG-azo polymers. OLZ, Olsalazine; EDC,1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; PCL, polycaprolactone.
drug dimethyl fumarate (DMF) (5 mg) was dissolved in acetone, followed by mixing with the polymer solutions. To remove the free DMF, the obtained DMF-loaded micelles were separated and purified for four times by repeating ultra-centrifugation (8500g for 15 min) and dispersion cycle in deionized water. 2.4. Characterization Fourier transform infrared spectra (FT-IR) were detected with FTIR spectrometer (spectrum 100, Perkin Elmer, USA).1H Nuclear Magnetic Resonance (1H NMR) spectra were measured by using Bruker AM 300 apparatus. The morphology of micelles was characterized by using transmission electron microscopy (TEM) (JEM1220, JEOL Ltd., Tokyo, Japan). Dynamic light scattering (DLS) (ZetaPALS, Brookhaven instruments corporation, U.S.A) was used to measure the mean diameter and polydispersity index of the micelles. 2.5. Dimethyl fumarate (DMF) measurement by HPLC DMF degradation rate and the release in different media were measured by using high efficiency liquid chromatography (HPLC) method (Waters, USA). The measurement conditions were: C18: 250 mm 4.6 mm 5 mm; UV measuring wavelength: 220 nm; mobile phase: water/acetonitrile (40:60); flow speed: 0.8 mL/ min; sample injection volume: 20 lL, the retention time of DMF was around 4.0 min. The equations of standard curves of DMF dissolved in deionized water and in the mixed solution of THF and water were Y (mg/L) = 1.334957e-5 X (Peak area) 2.8821 (R = 0.9971) and Y (mg/L) = 3.838324 e-5 X (Peak area) 0.23848 (R = 0.99964). 2.6. Measurement of DMF loading in micelles The procedure to measure DMF loading in micelles was as following: firstly, the concentration of free DMF was measured directly by using HPLC. Then, the micelles were destroyed with THF. A DMF-loaded micelle solution (2 mL) was poured in
volumetric flask before diluted with THF to 5 mL, the final solution was vibrated sharply and mixed homogeneously to destroy the micelles, and the total concentration of DMF in the micelles solution was measured. The concentration of DMF encapsulated in the micelles was the difference value between the total concentration of DMF after the micelles destroyed and that of DMF free from the micelles. The drug loading content was calculated as following equation:
Percentage of drug loading ¼ ððWeight of drug in micellesÞ= ðWeight of micellesÞÞ 100%; Encapsulation Efficiency ¼ ððWeight of drug encapsulated in micellesÞ= ðWeight of total drugÞÞ 100%:
2.7. DMF release from DMF-loaded micelles To measure the drug release in different medias, a drug-loading micelles solution (0.8 mL) was poured in volumetric flask before diluting with the corresponding media to 2 mL, the final solution was decanted into a 5 mL centrifuge tube and incubated in 37 °C incubator. Each tube was taken out at designed time to measure the concentration of DMF encapsulated in the micelles. The cumulative release rate was calculated as following equation: Cumulative release rate = (1 (weight of drugs encapsulated in micelles at designed time)/(weight of drugs encapsulated in micelles without incubation)) 100%. 2.8. Cell viability measurement Cell viability was measured by using the MTT assay, as described in our previous work [11]. Briefly, cells were seeded in 96-well flat-bottomed plates at 5000 cells/well. Different culture medium containing the tested drugs was added and incubated for 24 h, respectively. Normal cell culture medium (Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum) was as control. Then the medium was removed and basal
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medium containing MTT (0.5 mg/mL) was added and further incubated for 4 h at 37 °C. Medium was then aspirated and. DMSO (180 lL) was pipetted to dissolve the crystal. Absorbance was measured at a wavelength of 490 nm with a plate reader (Tecan Infinite m200, Mannedorf, Switzerland). 2.9. Live and dead staining The LIVE/DEADÒ Viability/Cytotoxicity Assay Kit (Invitrogen) was used to detect the live and dead cells as described in our previous work [11]. Briefly, cells were grown on coverslips at a density of 3.75 104 cells mL1 and incubated overnight at 37 °C in a humidified 5% CO2 incubator. The cells were washed with PBS and dyed according to the manufacturer’s instructions. The labeled cells were photographed under a fluorescence microscope. The live cells fluoresce green and dead cells fluoresce red. 2.10. Collection of rat gastric fluid and rat colonic fluid The adult male Sprague–Dawley rats (280–320 g) were purchased from Animal Center of Harbin Medical University. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University, P.R. China. The rats were anesthetized with 10% chloral hydrate. Dulbecco’s modified Eagle medium (DMEM) or phosphate buffered solution (PBS) was injected into the stomach or colon part after opening the animal abdominal cavity. Then the gastric or colonic fluid was extracted and collected into 15 mL centrifuge tubes. This procedure was repeated for three times. Finally, the tubes were centrifuged at 500g for 20 min, and the supernatants were filtered by 0.22 lm microporous membrane. For the DMF release experiments, the gastric and colonic fluids were harvested by using PBS; for the cell culture experiments, the colonic fluid was harvested by using DMEM and diluted 10 times.
Fig. 2. FT-IR spectra of sPCEG-azo polymers.
2.11. Statistical analysis Data were presented as mean ± standard error (SE). Significance was determined by using Student’s t-test or one-way ANOVA followed by Holm-Sidak. P < 0.05 was considered significant. 3. Results and discussion 3.1. Characterization of sPCEG-azo polymer and DMF-loaded sPCEGazo polymeric micelles The amphiphilic azo polymer sPCEG-azo was synthesized from the star-shape PCL and mPEG which were connected with OLZ. The chemical structure of sPCEG-azo was characterized with FT-IR and 1 H NMR methods. In the FT-IR spectra (Fig. 2), the absorption bond at 1728 cm1 was the C@O bond stretching vibrations which related to the eater bond of the star-shape PCL, the typical absorption bonds at 1242 cm1 and 1186 cm1 were attributed to the C– O–C and the –COO– stretching vibrations of mPEG respectively. Moreover, the absorption band at 1672 cm1 indicated that the aromatic acid ester bond existed in the azo polymer (Fig. 2). The 1H NMR spectra of sPCEG-azo and OLZ were shown in Fig. 3A and Fig. 3B respectively. In Fig. 3A, the typical peaks of PCL appeared at 1.35 ppm, 1.64 ppm, 2.29 ppm and 4.04 ppm which were represented by a, b + b0 , c, d, the characteristic peak of methylene protons of –CH2CH2O– appeared at 3.62 ppm where was labeled by h was assigned to mPEG, while the peak at 3.36 ppm which was labeled by i belonged to methoxy protons of mPEG, the inset of Fig. 3A was a blown up of 1H NMR spectra from 5 ppm to 13 ppm, the peaks at 7.08 ppm, 8.03 ppm,
Fig. 3. 1H NMR spectra of sPCEG-azo polymers (A) and OLZ (B). OLZ, Olsalazine.
8.48 ppm marked e, f and g were corresponding with the peaks of 2, 3, 4 in Fig. 3B which exhibited the 1H NMR spectra of OLZ, while the weak peak marked j in the inset of Fig. 3A belonging to the phenolic hydroxyl protons of OLZ was shifted comparing with the corresponding peak in Fig. 3B, indicating that the carboxyl group of OLZ participated in the esterification reaction, therefore, the 1H NMR spectra results further confirmed that the azo bond was introduced successfully. The electron microscope image of DMF-loaded sPCEG-azo polymeric micelles was shown in Fig. 4A and the dynamic light
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scattering (DLS) analysis was shown in Fig. 4B. The DLS results showed that the DMF-loaded sPCEG-azo polymeric micelles had a mean diameter of 153.6 nm and a polydispersity index of 0.195, confirming that DMF-loaded sPCEG-azo polymeric micelles had a narrow-size distribution. 3.2. DMF release from micelles Because of the trace solubility of DMF in water, a little amount of DMF out of the micelles would influence the quantitative analysis of DMF encapsulated in the micelles. Therefore, a certain amount of THF was added to destroy the micelles, and the net amount of DMF encapsulated in the micelles was calculated by subtracting the DMF dissolved in water. Fig. 5 showed the appearance of DMF-loaded sPCEG-azo micelles before and after THF treatment. The solution of sPCEG-azo micelles encapsulated with DMF was transparent, but extremely turbid when the micelles were destroyed by THF. We quantified DMF content by using HPLC method. We found that DMF degraded in normal PBS solution (Fig. 6A), therefore, the DMF was quantified by using THF disruption methods as mentioned above. The loading content of DMF in sPCEG-azo micelles was 5.32 ± 0.5% (mg/mg), and 4.8 ± 0.5% (mg/mg) in sPCEG micelles without azo bond; the encapsulation efficiency of DMF in sPCEG-azo micelles was 24.71 ± 0.5% and 24.8 ± 0.5% of azofree micelles, indicating that the loading capacity was similar in polymers containing azo bond or not. The loading content of DMF in sPCEG-azo micelles was 4.7 ± 0.5% (mg/mg) after two weeks at 4 °C, which was not significantly reduced, indicating that sPCEG-azo micelles was stable. In order to verify the azo enzyme-responsive cleavage of the azo bond in the sPCEG-azo micelles and the colon-specific targeting of sPCEG-azo micelles, we compared the DMF release from sPCEG and sPCEG-azo micelles in different experimental conditions. As shown in Fig. 6B, the cumulative release of DMF from sPCEG micelles in PBS (pH 7.4), RGF (pH 1.2) and RCF (pH 7.4) was no more than 20% during the first 10 h, indicating that the degradation of sPCEG micelles was not sensitive to the gastric or colonic fluids. With the time extending from 12 h to 24 h, the cumulative release of DMF from sPCEG micelles in PBS and RCF was gradually increased, indicating that, to a certain extent, there seemed to be a DMF release via diffusion fashion. However, it should be emphasized that, although DMF might be slowly released with a diffusion fashion from the micelles, the release was not significant in the first 10 h (Fig. 6B). Compared with that in PBS or rat gastric fluid, the cumulative release of DMF from sPCEG-azo micelles was significantly acceler-
Fig. 5. The photographs show that THF treatment disrupted the micelles and DMF was released. THF, tetrahydrofuran.
ated in the rat colonic fluid (Fig. 6C). The DMF release from sPCEGazo micelles in rat colonic fluid was 67.20% in the first 1 h, and almost 100% within 10 h (Fig. 6C). In the rat gastric fluid, the cumulative release of DMF from sPCEG-azo micelles was no more than 20% during the first 10 h (Fig. 6C), similar to that of sPCEG micelles. These results indicated that the sPCEG-azo micelles were stable in stomach and the azo bond from OLZ in the polymers play a critical role in the rapid DMF release from the DMF-loaded sPCEG-azo micelles. These properties of DMF-loaded sPCEG-azo micelles could result in a higher DMF concentration in colon part and meet the demand of colon-targeted drug delivery. We further observed the state of DMF-loaded sPCEG-azo polymer micelles solution and examined the morphology of DMFloaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids by using digital photography and TEM. In PBS solution and rat gastric fluid for 24 h at 37 °C, the transparency of DMF-loaded sPCEG-azo micelles was not changed (Fig. 7A (a and b)), however, in rat colon fluid for 24 h at 37 °C, the solution of DMF-loaded sPCEG-azo micelles became significantly turbid, which was due to the azo bond in the micelles cleaved by azo enzyme in the colon fluid, thus the hydrophobic groups separated from hydrophilic groups reunited and DMF was released. As shown in Fig. 7B (a and b) of TEM images, the DMF-loaded sPCEG-azo micelles kept integrated micelles shape in PBS and rat gastric fluids (pH 1.2) at 37 °C for 24 h, however, correspondingly, most of the DMFloaded sPCEG-azo micelles clustered in rat colonic fluids (pH 7.4). As shown in the enlarged frame of Fig. 7B (c), some micelles fused to form a larger cluster, which was consistent with the observation by digital photography and explained the turbidity phenomena in Fig. 7A (c).
Fig. 4. Transmission electron microscopic (TEM) image (A) and dynamic light scattering (DLS) analysis of DMF-loaded sPCEG-azo polymer micelles (B).
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Fig. 6. DMF release from sPCEG-azo and sPCEG micelles in PBS, rat gastric and colonic fluid. (A) The degradation curve of DMF in PBS solution. (B) DMF release from sPCEG micelles in PBS, rat gastric and colonic fluids. (C) DMF release from DMF-loaded sPCEG-azo micelles in PBS, rat gastric and colonic fluids. PBS, phosphate buffered solution; RGF, rat gastric fluid; RCF, rat colonic fluid. The experiments were repeated three times.
Fig. 7. Photographs and TEM images of DMF-loaded sPCEG-azo polymeric micelles in PBS, rat gastric and colonic fluids. (A) The photographs of DMF-loaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids at 0 and 24 h. It was apparent that the micelle solution became turbid in rat colonic fluid after 24 h. (B) The TEM images of DMF-loaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids (24 h). TEM, Transmission electron microscopy. PBS, phosphate buffered solution; RGF, rat gastric fluid; RCF, rat colonic fluid.
3.3. Cytotoxic effects of the DMF-loaded micelles In order to identify that DMF was loaded inside the micelles, we examined the cytotoxic effects of the DMF-loaded sPCEG-azo polymer micelles on colon cancer cell lines CT26, HT29, and HCT116 cells, compared with the equivalent amount of DMF (100 lM). As shown in Fig. 8A, the cell viability assay showed that the DMFloaded sPCEG-azo polymer micelles (equivalent to 100 lM DMF) had no significant cytotoxic effect on HCT116 and HT29 cells in normal cell culture conditions, except a slight cytotoxic effect on CT26 cells; however, the equivalent amount of DMF (100 lM) significantly inhibited the cell viability of CT26, HT29, and HCT116 cells. Furthermore, the LIVE/DEADÒ Viability/Cytotoxicity assay showed that DMF (100 lM) significantly reduced the number of green cells and increased the number of red cells (the live cells fluoresce green and dead cells fluoresce red), but the micelles loaded with equivalent amount of DMF had no significant effects (Fig. 8B).
These results indicated that the DMF-loaded sPCEG-azo polymer micelles were successfully assembled. Next, we examined the cytotoxic effect of the DMF-loaded sPCEG-azo polymeric micelles on CT26 and HCT116 colon cancer cells cultured in rat colonic fluid. Compared with the normal cell culture conditions, the cell viability of CT26 and HCT116 cells was relatively lower (Fig. 9A and B). Because that the components in RCF were different with that in normal culture media, it was reasonable that the cell viability of CT26 and HCT116 cells was relatively lower. Compared with the conditions in rat colonic fluid, the cell viability of CT26 and HCT116 cells treated with DMFloaded sPCEG-azo polymeric micelles was significantly reduced, similarly to that of DMF treatment (Fig. 9A and B). The blank polymer micelles showed no significant cytotoxic effect on CT26 and HCT116 cells in rat colonic fluid culture conditions. We further examined the cytotoxic effects of DMF-loaded sPCEG polymeric micelles (azo-free) on CT26 and HCT116 cells.
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Fig. 8. The cytotoxic effect of the DMF-loaded sPCEG-azo polymeric micelles on CT26, HT29, and HCT116 colon cancer cells in normal culture medium. (A) The cell viability assay showed that the DMF-loaded sPCEG-azo polymeric micelles with DMF (100 lM) had no significant cytotoxic effects on CT26, HT29, and HCT116 cells; however, the equivalent amount of DMF (100 lM) significantly inhibited the cell viability of CT26, HT29, and HCT116 cells. **P < 0.01 vs control. n = 24 in each group. (B) The LIVE/DEADÒ Viability/Cytotoxicity staining images showed that DMF (100 lM) but not the DMF-loaded sPCEG-azo polymeric micelles with equivalent amount of DMF reduced the live cells and increased the dead cells. The live cells fluoresce green and dead cells fluoresce red. The cell viability was measured after 24 h treatment.
Fig. 9. The cytotoxic effect of the DMF-loaded micelles on CT26 and HCT116 colon cancer cells in rat colonic fluid. The cell viability was measured after 24 h treatment. ** P < 0.01 vs RCF. The original rat colonic fluid (RCF) was diluted 10 times to culture cells. Control is in normal cell culture conditions. The concentration of DMF was 100 lM; The content of DMF amount in DMF-loaded micelles was equivalent to 100 lM. RCF, rat colonic fluid. DMF, dimethyl fumarate. DMF-loaded micelles, DMF-loaded sPCEG-azo polymeric micelles. Blank micelles, sPCEG-azo polymeric micelles without DMF.
In both normal cell culture and RCF culture conditions, the azo-free micelles (equivalent to 100 lM DMF) showed significant cytotoxic effect (Fig. 10), which was in line with the data of DMF cumulative
release in Fig. 6. We also noticed that, compared with DMF-loaded sPCEG-azo polymeric micelles (Fig. 8A), the azo-free micelles showed more strong cytotoxic effect (Fig. 10), which might be
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DMF as a strategy for colon-targeted chemotherapy. Our design is a double-win strategy, the advantages include: (1) DMF is locally released in colon part and the local high concentration of DMF induces necroptosis of colon cancer cells and circumvents cancer drug resistance.(2) DMF is metabolized into MMF after absorption, which decreases the systemic toxicity. (3) DMF-included necroptosis might lead to inflammation which is ameliorated by 5-aminosalicylic acid from OLZ, thus their pharmacological effects are mutual complementary. A recent work synthesized a azobenzene-linked poly (ethylene glycol)-b-poly(styrene) (PEG-N = N-PS) amphiphilic copolymer and attempted to use it to establish a colon-specific delivery system [18]. Their study shares the same hypotheses with us, but presently, we have applied this system to deliver a certain drug DMF, which has been proven to inhibit colon cancer cells in our previous work [11]. The present study provides a novel chemotherapeutic strategy for colon cancer treatment. Disclosure statement None. Fig. 10. The cytotoxic effect of DMF-loaded sPCEG polymeric micelles (azo-free) on CT26 and HCT116 cells. The cell viability was measured after 24 h treatment. ** P < 0.01 vs control or RCF. RCF, rat colonic fluid; Azo-free, DMF-loaded sPCEG polymeric micelles.
due to the difference of drug release property. We did not elucidate the exact mechanisms, which was the limitation of the present study. Presently, the major colon-targeted drug delivery approaches via oral route include: (1) temporal control of drug delivery (depends on time of passage); (2) pH-based drug delivery (triggered by a change in local pH); (3) enzyme-based drug delivery; (4) pressure-based systems [15]. Other novel therapies include receptor (like epidermal growth factor receptor, folate receptor, VEGF receptor, hyaluronic acid receptor) based targeting therapy, colon targeted proapoptotic anticancer drug delivery system, and gene therapy [15]. Base on the above principles, several new techniques were applied recently, for instance, a modified tri-axial electrospinning process was developed for the generation of a new type of pH-sensitive polymer/lipid nanocomposite to provide a colon-targeted sustained release [16]. We designed the DMFloaded sPCEG-azo polymeric micelles based on azoreductase specifically secreted by colonic bacteria. However, the most novel point of the present work was that we took advantage of the pharmacological properties of olsalazine and dimethyl fumarate and established a technical platform for colon-targeted drug delivery, which was illuminating in this research field.
4. Conclusions Olsalazine is an effective oral treatment for both active ulcerative colitis and for maintenance of disease remission. As a prodrug, OLZ is a dimer of 5-aminosalicylic acid, which is linked by the azo bond. When taken orally, OLZ is less absorbed in stomach and small intestine, but in the colon part, the azo bond in the structure is cleaved by azoreductase produced by the microbial flora present in the colon, and 5-aminosalicylic acid is liberated. 5aminosalicylic acid is the component of OLZ for therapy of inflammatory bowel diseases, it has a selective positive effect on ulcerative colitis in inducing remission, preventing relapse and possibly reducing the risk of cancer [17]. Since the azo bond exists in the OLZ structure, OLZ itself can be used as biomaterials with colontargeted properties triggered by colon microbial flora. Based on this hypothesis, we design OLZ-linked polymer micelles containing
Author contributions Zhen-Gang Ma, Rui Ma, and Yu-Feng Zheng synthesized and characterized the polymers and micelles. Xiao-Lin Xiao, Yong-Hui Zhang, Xin-Zi Zhang, Nan Hu, Jin-Lai Gao, and De-Li Dong performed the cell culture and cytotoxic experiments. Zhi-Jie Sun designed the project and wrote the paper. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (HEUCF201310020) from Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education, PR China. References [1] W.A. Hammond, A. Swaika, K. Mody, Pharmacologic resistance in colorectal cancer: a review, Ther. Adv. Med. Oncol. 8 (2016) 57–84. [2] S. Amidon, J.E. Brown, V.S. Dave, Colon-targeted oral drug delivery systems: design trends and approaches, AAPS PharmSciTech. 16 (2015) 731–741. [3] S. Fulda, Therapeutic exploitation of necroptosis for cancer therapy, Semin. Cell Dev. Biol. 35 (2014) 51–56. [4] L. Steinhart, K. Belz, S. Fulda, Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis, Cell Death Dis. 12 (2013) e802. [5] W. Han, L. Li, S. Qiu, Q. Lu, Q. Pan, Y. Gu, J. Luo, X. Hu, Shikonin circumvents cancer drug resistance by induction of a necroptotic death, Mol. Cancer Ther. 6 (2007) 1641–1649. [6] E. Grassilli, R. Narloch, E. Federzoni, L. Ianzano, F. Pisano, R. Giovannoni, G. Romano, L. Masiero, B.E. Leone, S. Bonin, M. Donada, G. Stanta, K. Helin, M. Lavitrano, Inhibition of GSK3B bypass drug resistance of p53-null colon carcinomas by enabling necroptosis in response to chemotherapy, Clin. Cancer Res. 19 (2013) 3820–3831. [7] C.B. Burness, E.D. Deeks, Dimethyl fumarate: a review of its use in patients with relapsing-remitting multiple sclerosis, CNS Drugs 28 (2014) 373–387. [8] R.J. Fox, M. Kita, S.L. Cohan, L.J. Henson, J. Zambrano, R.H. Scannevin, BG-12 (dimethyl fumarate): a review of mechanism of action, efficacy, and safety, Curr. Med. Res. Opin. 30 (2014) 251–262. [9] R. Loewe, T. Valero, S. KremLing, B. Pratscher, R. Kunstfeld, H. Pehamberger, P. Petzelbauer, Dimethylfumarate impairs melanoma growth and metastasis, Cancer Res. 66 (2006) 11888–11896. [10] W.G. Kirlin, J. Cai, M.J. DeLong, E.J. Patten, D.P. Jones, Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells, J. Nutr. 129 (1999) 1827–1835. [11] X. Xie, Y. Zhao, C.Y. Ma, X.M. Xu, Y.Q. Zhang, C.G. Wang, J. Jin, X. Shen, J.L. Gao, N. Li, Z.J. Sun, D.L. Dong, Dimethyl fumarate induces necroptosis in colon cancer cells through GSH depletion/ROS increase/MAPKs activation pathway, Br. J. Pharmacol. 172 (2015) 3929–3943. [12] C. Lin, H.L. Ng, W. Pan, H. Chen, G. Zhang, Z. Bian, A. Lu, Z. Yang, Exploring different strategies for efficient delivery of colorectal cancer therapy, Int. J. Mol. Sci. 16 (2015) 26936–26952.
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[16] C. Yang, D.G. Yu, D. Pan, X.K. Liu, X. Wang, S.W. Bligh, G.R. Williams, Electrospun pH-sensitive core-shell polymer nanocomposites fabricated using a tri-axial process, Acta Biomater. 35 (2016) 77–86. [17] Ø. Hauso, T.C. Martinsen, H. Waldum, 5-Aminosalicylic acid, a specific drug for ulcerative colitis, Scand. J. Gastroenterol. 50 (2015) 933–941. [18] J. Rao, A. Khan, Enzyme sensitive synthetic polymer micelles based on the azobenzene motif, J. Am. Chem. Soc. 135 (2013) 14056–14059.
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Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy Zhen-Gang Ma a, Rui Ma a, Xiao-Lin Xiao b,c, Yong-Hui Zhang b,c, Xin-Zi Zhang b,c, Nan Hu b,c, Jin-Lai Gao b,c, Yu-Feng Zheng a, De-Li Dong b,c, Zhi-Jie Sun a,⇑ a
Institute of Materials Processing and Intelligent Manufacturing & Center for Biomedical Materials and Engineering, Harbin Engineering University, PR China Department of Pharmacology (the State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education), College of Pharmacy, Harbin Medical University, PR China c Translational Medicine Research and Cooperation Center of Northern China, Heilongjiang Academy of Medical Sciences, Harbin Medical University, PR China b
a r t i c l e
i n f o
Article history: Received 26 April 2016 Received in revised form 10 August 2016 Accepted 16 August 2016 Available online xxxx Keywords: Colon-targeted Dimethyl fumarate Micelles Olsalazine Colon cancer cells
a b s t r a c t Colon-targeted drug delivery and circumventing drug resistance are extremely important for colon cancer chemotherapy. Our previous work found that dimethyl fumarate (DMF), the approved drug by the FDA for the treatment of multiple sclerosis, exhibited anti-tumor activity on colon cancer cells. Based on the pharmacological properties of DMF and azo bond in olsalazine chemical structure, we designed azo polymeric micelles for colon-targeted dimethyl fumarate delivery for colon cancer therapy. We synthesized the star-shape amphiphilic polymer with azo bond and fabricated the DMF-loaded azo polymeric micelles. The four-arm polymer star-PCL-azo-mPEG (sPCEG-azo) (constituted by star-shape PCL (polycaprolactone) and mPEG (methoxypolyethylene glycols)-olsalazine) showed self-assembly ability. The average diameter and polydispersity index of the DMF-loaded sPCEG-azo polymeric micelles were 153.6 nm and 0.195, respectively. In vitro drug release study showed that the cumulative release of DMF from the DMF-loaded sPCEG-azo polymeric micelles was no more than 20% in rat gastric fluid within 10 h, whereas in the rat colonic fluids, the cumulative release of DMF reached 60% in the initial 2 h and 100% within 10 h, indicating that the DMF-loaded sPCEG-azo polymeric micelles had excellent colon-targeted property. The DMF-loaded sPCEG-azo polymeric micelles had no significant cytotoxicity on colon cancer cells in phosphate buffered solution (PBS) and rat gastric fluid. In rat colonic fluid, the micelles showed significant cytotoxic effect on colon cancer cells. The blank sPCEG-azo polymeric micelles (without DMF) showed no cytotoxic effect on colon cancer cells in rat colonic fluids. In conclusion, the DMF-loaded sPCEG-azo polymeric micelles show colon-targeted DMF release and anti-tumor activity, providing a novel approach potential for colon cancer therapy. Statement of Significance Colon-targeted drug delivery and circumventing drug resistance are extremely important for colon cancer chemotherapy. Our previous work found that dimethyl fumarate (DMF), the approved drug by the FDA for the treatment of multiple sclerosis, exhibited anti-tumor activities on colon cancer cells (Br J Pharmacol. 2015 172(15):3929-43.). Based on the pharmacological properties of DMF and azo bond in olsalazine chemical structure, we designed azo polymeric micelles for colon-targeted dimethyl fumarate delivery for colon cancer therapy. We found that the DMF-loaded sPCEG-azo polymeric micelles showed colon-targeted DMF release and anti-tumor activities, providing a novel approach potential for colon cancer therapy. Ó 2016 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
Abbreviations: DMF, dimethyl fumarate; OLZ, olsalazine; PCL, polycaprolactone; mPEG, methoxypolyethylene glycols; EDC, 1-(3-dimethylaminopropyl)-3-ethylcar bodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; sPCEG, star-PCLmPEG; sPCEG-azo, star-PCL-azo-mPEG; TEM, transmission electron microscopy. ⇑ Corresponding author at: Institute of Materials Processing and Intelligent Manufacturing, Center for Biomedical Materials and Engineering, Harbin Engineering University, 145 Nantong Street, Nangang District, Harbin 150001, PR China. E-mail address: [email protected] (Z.-J. Sun).
1. Introduction Colon cancer is one of the most prevalent diseases, and remains a significant cause of morbidity and mortality worldwide. Despite advances in chemotherapy, resistance to the anti-cancer drugs is still the greatest challenge in the management of colon cancer.
http://dx.doi.org/10.1016/j.actbio.2016.08.021 1742-7061/Ó 2016 Published by Elsevier Ltd on behalf of Acta Materialia Inc.
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The main drugs for chemotherapy of colon cancer include 5-fluorouracil (5-FU), capecitabine, tegafur, irinotecan, and oxaliplatin etc. However, resistance to these chemotherapies has arisen and the molecular mechanisms include decreased intracellular drug concentration, altered metabolism, or alterations of targets for the therapy [1]. Recent studies suggest that inducing necroptosis of colon cancer cells and colon-targeted drug delivery could increase the efficacy of anticancer drugs and decrease the drug resistance [2–4]. Necroptosis is a type of cell death different with apoptosis [5]. Necroptosis is characterized with (1) a morphology of necrotic cell death; (2) loss of plasma membrane integrity; (3) loss of mitochondrial membrane potentials; (4) elevation of reactive oxygen species; and (5) that the cell death was prevented by a small molecule, necrostatin-1 [5]. Han et al. proved that inducing necroptosis had a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance [5]. Grassilli et al. reported that, in drug-resistant colon carcinoma cells, induction of necroptosis by GSK3B silencing resensitized drug-resistant cells to chemotherapy [6]. Therefore, induction of necroptosis would be an approach to circumvent cancer drug resistance. Dimethyl fumarate (DMF) is the methyl ester of fumaric acid and has been approved by the FDA and European Medicines Agency as a new oral drug for the treatment of multiple sclerosis [7,8]. DMF also exhibits anti-tumor effects, for instance, it inhibits melanoma growth and metastasis [9] and induces apoptosis in HT29 colon carcinoma cells [10]. Our previous work found that DMF but not its metabolite MMF induced necroptosis in colon cancer cells through a mechanism involving the depletion of GSH, increase of ROS and activation of MAPKs [11], indicating that DMF would be a necroptosis inducer which circumvent colon cancer drug resistance. However, it should be noted that DMF could be easily hydrolyzed to mono-methylfumarate (MMF) which showed no cytotoxic effect on colon cancer cells [11]. Therefore, it must be avoided that DMF was hydrolyzed in stomach and small intestine if DMF was attempted to be developed as a local chemotherapeutic drug for colon cancer. Presently, several strategies for colon-specific drug delivery have been explored, including the techniques dependent on pH, time, pressure and/or bacteria [12]. Colonic microfloras consist of anaerobic bacteria that are only present in the colon region and secrete specific biodegradable enzymes, this feature can be exploited to target colonic drug release. Olsalazine (OLZ) is a dimer of 5-aminosalicylic acid that are linked via an azo bond. When OLZ reaches the colon part, it is cleaved by azoreductase specifically secreted by colonic bacteria [12]. Azo bond in OLZ structure indicates that OLZ can be used as a bacteria-triggered, colon-targeted materials. Therefore, in the present study, we design DMF-loaded OLZ-linked polymer micelles (Fig. 1A) and investigate the DMF release and anti-tumor activity of these micelles. Our work will provide a novel approach for colon cancer therapy.
product olsalazine was dried with vacuum at 60 °C. All other chemicals were purchased from commercial supplier.
2. Materials and methods
2.3. Assembly of polymeric micelles
2.1. Materials
The blank micelles of sPCEG-azo and sPCEG polymers were fabricated by the evaporation method. In brief, the polymer solution containing 20 mg sPCEG-azo or sPCEG in 20 mL tetrahydrofuran (THF) was added dropwisely into 40 mL deionized water with the rotor stirring. The micelle solution was filtered by 0.45 lm organic filter membrane after evaporating off THF at room temperature. The method to fabricate dimethyl fumarate (DMF)-loaded micelles was same as that for the blank micelles. The anticancer
e-Caprolactone (e-Cl), erythritol, and all the catalysts were purchased from Aladdin@ company (Shanghai, China). mPEG (Methoxypolyethylene glycols) (Mn = 2 kDa) was purchased from Energy Chemical. Co. Ltd (Shanghai, China), Olsalazine Sodium (OLZ-Na) was purchased from Jusheng Technology. Co. Ltd (Hubei, China). OLZ-Na was acidized with hydrochloric acid and the
2.2. Synthesis procedures 2.2.1. Synthesis of the star-shape PCL (polycaprolactone) The star-shape PCL was synthesized by ring-opening polymerization as described in previous work with modification [13]. e-Cl was purified by reduced pressure distillation after dried with 4A molecular sieve and added into a 100 mL round-bottom flask followed by adding Sn(oct)2 as catalyst and erythritol as initiator. The flask together with its content was degassed under vacuum for 5 h at room temperature, then the ring-opening polymerization reaction was processed at 140 °C for 6 h. After the reaction system cooled down, the products were dissolved in dichloromethane (DCM) and precipitated with cold ethyl alcohol before filtration. Finally, the star-shape PCL polymers were obtained after vacuum drying for 24 h. 2.2.2. Synthesis of methoxypolyethylene-olsalazine (mPEG-OLZ) The polymerization procedures were as following: mPEG and excess olsalazine (OLZ) were dissolved in N,N-dimethylformamide followed by adding 1-(3-dimethylaminopropyl)-3-ethylcarbodii mide hydrochloride (EDC) and 4-dimethylaminopyridine (DMAP) as catalysts into the reaction system, the reaction processed at 35 °C for 48 h. Then, the product was precipitated with cold diethyl ether before filtration and redissolved in N, N-dimethylformamide, the solution was transferred into a dialysis tube (MWCO = 1 kDa) and immersed in deionized water to remove the excess catalysts and the un-reacted OLZ. Finally, the mPEG-OLZ polymers were obtained after lyophilized. 2.2.3. Synthesis of mPEG-COOH The mPEG-COOH was synthesized as described in previous work [14]. In brief, mPEG, butanedioic anhydride and the catalyst DMAP were added in a two-neck round-bottom flask and dissolved in dichloromethane, then the reaction processed at room temperature for 24 h. Next, the reactant solution was precipitated with cold diethyl ether before filtration. Finally, the products were obtained after vacuum drying for 24 h. 2.2.4. Synthesis of star-PCL-azo-mPEG (sPCEG-azo) and star-PCLmPEG (sPCEG) To synthesize the polymers with azo linkage or without azo linkage, the star-shape PCL and excess mPEG-OLZ or mPEGCOOH were dissolved in N,N-dimethylformamide followed by adding EDC and DMAP. The reaction processed at 35 °C for 48 h. Then, the reactant solution was precipitated with cold diethyl ether before filtration, and the product was redissolved in N,N-dimethylformamide. The solution was transferred into dialysis tube (MWCO = 3.5 kDa) to remove the unreacted reactants, catalysts, and N,N-dimethylformamide. Finally, the star-PCL-azomPEG (sPCEG-azo) polymers and star-PCL-mPEG (sPCEG) polymers were obtained after lyophilized. The total synthesis routes of the star-shape amphiphilic polymers were shown in Fig. 1B.
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Fig. 1. (A) The design schematic diagram of the project. DMF, dimethyl fumarate. (B) The total synthesis routes of sPCEG-azo polymers. OLZ, Olsalazine; EDC,1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP, 4-dimethylaminopyridine; PCL, polycaprolactone.
drug dimethyl fumarate (DMF) (5 mg) was dissolved in acetone, followed by mixing with the polymer solutions. To remove the free DMF, the obtained DMF-loaded micelles were separated and purified for four times by repeating ultra-centrifugation (8500g for 15 min) and dispersion cycle in deionized water. 2.4. Characterization Fourier transform infrared spectra (FT-IR) were detected with FTIR spectrometer (spectrum 100, Perkin Elmer, USA).1H Nuclear Magnetic Resonance (1H NMR) spectra were measured by using Bruker AM 300 apparatus. The morphology of micelles was characterized by using transmission electron microscopy (TEM) (JEM1220, JEOL Ltd., Tokyo, Japan). Dynamic light scattering (DLS) (ZetaPALS, Brookhaven instruments corporation, U.S.A) was used to measure the mean diameter and polydispersity index of the micelles. 2.5. Dimethyl fumarate (DMF) measurement by HPLC DMF degradation rate and the release in different media were measured by using high efficiency liquid chromatography (HPLC) method (Waters, USA). The measurement conditions were: C18: 250 mm 4.6 mm 5 mm; UV measuring wavelength: 220 nm; mobile phase: water/acetonitrile (40:60); flow speed: 0.8 mL/ min; sample injection volume: 20 lL, the retention time of DMF was around 4.0 min. The equations of standard curves of DMF dissolved in deionized water and in the mixed solution of THF and water were Y (mg/L) = 1.334957e-5 X (Peak area) 2.8821 (R = 0.9971) and Y (mg/L) = 3.838324 e-5 X (Peak area) 0.23848 (R = 0.99964). 2.6. Measurement of DMF loading in micelles The procedure to measure DMF loading in micelles was as following: firstly, the concentration of free DMF was measured directly by using HPLC. Then, the micelles were destroyed with THF. A DMF-loaded micelle solution (2 mL) was poured in
volumetric flask before diluted with THF to 5 mL, the final solution was vibrated sharply and mixed homogeneously to destroy the micelles, and the total concentration of DMF in the micelles solution was measured. The concentration of DMF encapsulated in the micelles was the difference value between the total concentration of DMF after the micelles destroyed and that of DMF free from the micelles. The drug loading content was calculated as following equation:
Percentage of drug loading ¼ ððWeight of drug in micellesÞ= ðWeight of micellesÞÞ 100%; Encapsulation Efficiency ¼ ððWeight of drug encapsulated in micellesÞ= ðWeight of total drugÞÞ 100%:
2.7. DMF release from DMF-loaded micelles To measure the drug release in different medias, a drug-loading micelles solution (0.8 mL) was poured in volumetric flask before diluting with the corresponding media to 2 mL, the final solution was decanted into a 5 mL centrifuge tube and incubated in 37 °C incubator. Each tube was taken out at designed time to measure the concentration of DMF encapsulated in the micelles. The cumulative release rate was calculated as following equation: Cumulative release rate = (1 (weight of drugs encapsulated in micelles at designed time)/(weight of drugs encapsulated in micelles without incubation)) 100%. 2.8. Cell viability measurement Cell viability was measured by using the MTT assay, as described in our previous work [11]. Briefly, cells were seeded in 96-well flat-bottomed plates at 5000 cells/well. Different culture medium containing the tested drugs was added and incubated for 24 h, respectively. Normal cell culture medium (Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum) was as control. Then the medium was removed and basal
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medium containing MTT (0.5 mg/mL) was added and further incubated for 4 h at 37 °C. Medium was then aspirated and. DMSO (180 lL) was pipetted to dissolve the crystal. Absorbance was measured at a wavelength of 490 nm with a plate reader (Tecan Infinite m200, Mannedorf, Switzerland). 2.9. Live and dead staining The LIVE/DEADÒ Viability/Cytotoxicity Assay Kit (Invitrogen) was used to detect the live and dead cells as described in our previous work [11]. Briefly, cells were grown on coverslips at a density of 3.75 104 cells mL1 and incubated overnight at 37 °C in a humidified 5% CO2 incubator. The cells were washed with PBS and dyed according to the manufacturer’s instructions. The labeled cells were photographed under a fluorescence microscope. The live cells fluoresce green and dead cells fluoresce red. 2.10. Collection of rat gastric fluid and rat colonic fluid The adult male Sprague–Dawley rats (280–320 g) were purchased from Animal Center of Harbin Medical University. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Harbin Medical University, P.R. China. The rats were anesthetized with 10% chloral hydrate. Dulbecco’s modified Eagle medium (DMEM) or phosphate buffered solution (PBS) was injected into the stomach or colon part after opening the animal abdominal cavity. Then the gastric or colonic fluid was extracted and collected into 15 mL centrifuge tubes. This procedure was repeated for three times. Finally, the tubes were centrifuged at 500g for 20 min, and the supernatants were filtered by 0.22 lm microporous membrane. For the DMF release experiments, the gastric and colonic fluids were harvested by using PBS; for the cell culture experiments, the colonic fluid was harvested by using DMEM and diluted 10 times.
Fig. 2. FT-IR spectra of sPCEG-azo polymers.
2.11. Statistical analysis Data were presented as mean ± standard error (SE). Significance was determined by using Student’s t-test or one-way ANOVA followed by Holm-Sidak. P < 0.05 was considered significant. 3. Results and discussion 3.1. Characterization of sPCEG-azo polymer and DMF-loaded sPCEGazo polymeric micelles The amphiphilic azo polymer sPCEG-azo was synthesized from the star-shape PCL and mPEG which were connected with OLZ. The chemical structure of sPCEG-azo was characterized with FT-IR and 1 H NMR methods. In the FT-IR spectra (Fig. 2), the absorption bond at 1728 cm1 was the C@O bond stretching vibrations which related to the eater bond of the star-shape PCL, the typical absorption bonds at 1242 cm1 and 1186 cm1 were attributed to the C– O–C and the –COO– stretching vibrations of mPEG respectively. Moreover, the absorption band at 1672 cm1 indicated that the aromatic acid ester bond existed in the azo polymer (Fig. 2). The 1H NMR spectra of sPCEG-azo and OLZ were shown in Fig. 3A and Fig. 3B respectively. In Fig. 3A, the typical peaks of PCL appeared at 1.35 ppm, 1.64 ppm, 2.29 ppm and 4.04 ppm which were represented by a, b + b0 , c, d, the characteristic peak of methylene protons of –CH2CH2O– appeared at 3.62 ppm where was labeled by h was assigned to mPEG, while the peak at 3.36 ppm which was labeled by i belonged to methoxy protons of mPEG, the inset of Fig. 3A was a blown up of 1H NMR spectra from 5 ppm to 13 ppm, the peaks at 7.08 ppm, 8.03 ppm,
Fig. 3. 1H NMR spectra of sPCEG-azo polymers (A) and OLZ (B). OLZ, Olsalazine.
8.48 ppm marked e, f and g were corresponding with the peaks of 2, 3, 4 in Fig. 3B which exhibited the 1H NMR spectra of OLZ, while the weak peak marked j in the inset of Fig. 3A belonging to the phenolic hydroxyl protons of OLZ was shifted comparing with the corresponding peak in Fig. 3B, indicating that the carboxyl group of OLZ participated in the esterification reaction, therefore, the 1H NMR spectra results further confirmed that the azo bond was introduced successfully. The electron microscope image of DMF-loaded sPCEG-azo polymeric micelles was shown in Fig. 4A and the dynamic light
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scattering (DLS) analysis was shown in Fig. 4B. The DLS results showed that the DMF-loaded sPCEG-azo polymeric micelles had a mean diameter of 153.6 nm and a polydispersity index of 0.195, confirming that DMF-loaded sPCEG-azo polymeric micelles had a narrow-size distribution. 3.2. DMF release from micelles Because of the trace solubility of DMF in water, a little amount of DMF out of the micelles would influence the quantitative analysis of DMF encapsulated in the micelles. Therefore, a certain amount of THF was added to destroy the micelles, and the net amount of DMF encapsulated in the micelles was calculated by subtracting the DMF dissolved in water. Fig. 5 showed the appearance of DMF-loaded sPCEG-azo micelles before and after THF treatment. The solution of sPCEG-azo micelles encapsulated with DMF was transparent, but extremely turbid when the micelles were destroyed by THF. We quantified DMF content by using HPLC method. We found that DMF degraded in normal PBS solution (Fig. 6A), therefore, the DMF was quantified by using THF disruption methods as mentioned above. The loading content of DMF in sPCEG-azo micelles was 5.32 ± 0.5% (mg/mg), and 4.8 ± 0.5% (mg/mg) in sPCEG micelles without azo bond; the encapsulation efficiency of DMF in sPCEG-azo micelles was 24.71 ± 0.5% and 24.8 ± 0.5% of azofree micelles, indicating that the loading capacity was similar in polymers containing azo bond or not. The loading content of DMF in sPCEG-azo micelles was 4.7 ± 0.5% (mg/mg) after two weeks at 4 °C, which was not significantly reduced, indicating that sPCEG-azo micelles was stable. In order to verify the azo enzyme-responsive cleavage of the azo bond in the sPCEG-azo micelles and the colon-specific targeting of sPCEG-azo micelles, we compared the DMF release from sPCEG and sPCEG-azo micelles in different experimental conditions. As shown in Fig. 6B, the cumulative release of DMF from sPCEG micelles in PBS (pH 7.4), RGF (pH 1.2) and RCF (pH 7.4) was no more than 20% during the first 10 h, indicating that the degradation of sPCEG micelles was not sensitive to the gastric or colonic fluids. With the time extending from 12 h to 24 h, the cumulative release of DMF from sPCEG micelles in PBS and RCF was gradually increased, indicating that, to a certain extent, there seemed to be a DMF release via diffusion fashion. However, it should be emphasized that, although DMF might be slowly released with a diffusion fashion from the micelles, the release was not significant in the first 10 h (Fig. 6B). Compared with that in PBS or rat gastric fluid, the cumulative release of DMF from sPCEG-azo micelles was significantly acceler-
Fig. 5. The photographs show that THF treatment disrupted the micelles and DMF was released. THF, tetrahydrofuran.
ated in the rat colonic fluid (Fig. 6C). The DMF release from sPCEGazo micelles in rat colonic fluid was 67.20% in the first 1 h, and almost 100% within 10 h (Fig. 6C). In the rat gastric fluid, the cumulative release of DMF from sPCEG-azo micelles was no more than 20% during the first 10 h (Fig. 6C), similar to that of sPCEG micelles. These results indicated that the sPCEG-azo micelles were stable in stomach and the azo bond from OLZ in the polymers play a critical role in the rapid DMF release from the DMF-loaded sPCEG-azo micelles. These properties of DMF-loaded sPCEG-azo micelles could result in a higher DMF concentration in colon part and meet the demand of colon-targeted drug delivery. We further observed the state of DMF-loaded sPCEG-azo polymer micelles solution and examined the morphology of DMFloaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids by using digital photography and TEM. In PBS solution and rat gastric fluid for 24 h at 37 °C, the transparency of DMF-loaded sPCEG-azo micelles was not changed (Fig. 7A (a and b)), however, in rat colon fluid for 24 h at 37 °C, the solution of DMF-loaded sPCEG-azo micelles became significantly turbid, which was due to the azo bond in the micelles cleaved by azo enzyme in the colon fluid, thus the hydrophobic groups separated from hydrophilic groups reunited and DMF was released. As shown in Fig. 7B (a and b) of TEM images, the DMF-loaded sPCEG-azo micelles kept integrated micelles shape in PBS and rat gastric fluids (pH 1.2) at 37 °C for 24 h, however, correspondingly, most of the DMFloaded sPCEG-azo micelles clustered in rat colonic fluids (pH 7.4). As shown in the enlarged frame of Fig. 7B (c), some micelles fused to form a larger cluster, which was consistent with the observation by digital photography and explained the turbidity phenomena in Fig. 7A (c).
Fig. 4. Transmission electron microscopic (TEM) image (A) and dynamic light scattering (DLS) analysis of DMF-loaded sPCEG-azo polymer micelles (B).
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Fig. 6. DMF release from sPCEG-azo and sPCEG micelles in PBS, rat gastric and colonic fluid. (A) The degradation curve of DMF in PBS solution. (B) DMF release from sPCEG micelles in PBS, rat gastric and colonic fluids. (C) DMF release from DMF-loaded sPCEG-azo micelles in PBS, rat gastric and colonic fluids. PBS, phosphate buffered solution; RGF, rat gastric fluid; RCF, rat colonic fluid. The experiments were repeated three times.
Fig. 7. Photographs and TEM images of DMF-loaded sPCEG-azo polymeric micelles in PBS, rat gastric and colonic fluids. (A) The photographs of DMF-loaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids at 0 and 24 h. It was apparent that the micelle solution became turbid in rat colonic fluid after 24 h. (B) The TEM images of DMF-loaded sPCEG-azo polymer micelles in PBS, rat gastric and colonic fluids (24 h). TEM, Transmission electron microscopy. PBS, phosphate buffered solution; RGF, rat gastric fluid; RCF, rat colonic fluid.
3.3. Cytotoxic effects of the DMF-loaded micelles In order to identify that DMF was loaded inside the micelles, we examined the cytotoxic effects of the DMF-loaded sPCEG-azo polymer micelles on colon cancer cell lines CT26, HT29, and HCT116 cells, compared with the equivalent amount of DMF (100 lM). As shown in Fig. 8A, the cell viability assay showed that the DMFloaded sPCEG-azo polymer micelles (equivalent to 100 lM DMF) had no significant cytotoxic effect on HCT116 and HT29 cells in normal cell culture conditions, except a slight cytotoxic effect on CT26 cells; however, the equivalent amount of DMF (100 lM) significantly inhibited the cell viability of CT26, HT29, and HCT116 cells. Furthermore, the LIVE/DEADÒ Viability/Cytotoxicity assay showed that DMF (100 lM) significantly reduced the number of green cells and increased the number of red cells (the live cells fluoresce green and dead cells fluoresce red), but the micelles loaded with equivalent amount of DMF had no significant effects (Fig. 8B).
These results indicated that the DMF-loaded sPCEG-azo polymer micelles were successfully assembled. Next, we examined the cytotoxic effect of the DMF-loaded sPCEG-azo polymeric micelles on CT26 and HCT116 colon cancer cells cultured in rat colonic fluid. Compared with the normal cell culture conditions, the cell viability of CT26 and HCT116 cells was relatively lower (Fig. 9A and B). Because that the components in RCF were different with that in normal culture media, it was reasonable that the cell viability of CT26 and HCT116 cells was relatively lower. Compared with the conditions in rat colonic fluid, the cell viability of CT26 and HCT116 cells treated with DMFloaded sPCEG-azo polymeric micelles was significantly reduced, similarly to that of DMF treatment (Fig. 9A and B). The blank polymer micelles showed no significant cytotoxic effect on CT26 and HCT116 cells in rat colonic fluid culture conditions. We further examined the cytotoxic effects of DMF-loaded sPCEG polymeric micelles (azo-free) on CT26 and HCT116 cells.
Please cite this article in press as: Z.-G. Ma et al., Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.08.021
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Fig. 8. The cytotoxic effect of the DMF-loaded sPCEG-azo polymeric micelles on CT26, HT29, and HCT116 colon cancer cells in normal culture medium. (A) The cell viability assay showed that the DMF-loaded sPCEG-azo polymeric micelles with DMF (100 lM) had no significant cytotoxic effects on CT26, HT29, and HCT116 cells; however, the equivalent amount of DMF (100 lM) significantly inhibited the cell viability of CT26, HT29, and HCT116 cells. **P < 0.01 vs control. n = 24 in each group. (B) The LIVE/DEADÒ Viability/Cytotoxicity staining images showed that DMF (100 lM) but not the DMF-loaded sPCEG-azo polymeric micelles with equivalent amount of DMF reduced the live cells and increased the dead cells. The live cells fluoresce green and dead cells fluoresce red. The cell viability was measured after 24 h treatment.
Fig. 9. The cytotoxic effect of the DMF-loaded micelles on CT26 and HCT116 colon cancer cells in rat colonic fluid. The cell viability was measured after 24 h treatment. ** P < 0.01 vs RCF. The original rat colonic fluid (RCF) was diluted 10 times to culture cells. Control is in normal cell culture conditions. The concentration of DMF was 100 lM; The content of DMF amount in DMF-loaded micelles was equivalent to 100 lM. RCF, rat colonic fluid. DMF, dimethyl fumarate. DMF-loaded micelles, DMF-loaded sPCEG-azo polymeric micelles. Blank micelles, sPCEG-azo polymeric micelles without DMF.
In both normal cell culture and RCF culture conditions, the azo-free micelles (equivalent to 100 lM DMF) showed significant cytotoxic effect (Fig. 10), which was in line with the data of DMF cumulative
release in Fig. 6. We also noticed that, compared with DMF-loaded sPCEG-azo polymeric micelles (Fig. 8A), the azo-free micelles showed more strong cytotoxic effect (Fig. 10), which might be
Please cite this article in press as: Z.-G. Ma et al., Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.08.021
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DMF as a strategy for colon-targeted chemotherapy. Our design is a double-win strategy, the advantages include: (1) DMF is locally released in colon part and the local high concentration of DMF induces necroptosis of colon cancer cells and circumvents cancer drug resistance.(2) DMF is metabolized into MMF after absorption, which decreases the systemic toxicity. (3) DMF-included necroptosis might lead to inflammation which is ameliorated by 5-aminosalicylic acid from OLZ, thus their pharmacological effects are mutual complementary. A recent work synthesized a azobenzene-linked poly (ethylene glycol)-b-poly(styrene) (PEG-N = N-PS) amphiphilic copolymer and attempted to use it to establish a colon-specific delivery system [18]. Their study shares the same hypotheses with us, but presently, we have applied this system to deliver a certain drug DMF, which has been proven to inhibit colon cancer cells in our previous work [11]. The present study provides a novel chemotherapeutic strategy for colon cancer treatment. Disclosure statement None. Fig. 10. The cytotoxic effect of DMF-loaded sPCEG polymeric micelles (azo-free) on CT26 and HCT116 cells. The cell viability was measured after 24 h treatment. ** P < 0.01 vs control or RCF. RCF, rat colonic fluid; Azo-free, DMF-loaded sPCEG polymeric micelles.
due to the difference of drug release property. We did not elucidate the exact mechanisms, which was the limitation of the present study. Presently, the major colon-targeted drug delivery approaches via oral route include: (1) temporal control of drug delivery (depends on time of passage); (2) pH-based drug delivery (triggered by a change in local pH); (3) enzyme-based drug delivery; (4) pressure-based systems [15]. Other novel therapies include receptor (like epidermal growth factor receptor, folate receptor, VEGF receptor, hyaluronic acid receptor) based targeting therapy, colon targeted proapoptotic anticancer drug delivery system, and gene therapy [15]. Base on the above principles, several new techniques were applied recently, for instance, a modified tri-axial electrospinning process was developed for the generation of a new type of pH-sensitive polymer/lipid nanocomposite to provide a colon-targeted sustained release [16]. We designed the DMFloaded sPCEG-azo polymeric micelles based on azoreductase specifically secreted by colonic bacteria. However, the most novel point of the present work was that we took advantage of the pharmacological properties of olsalazine and dimethyl fumarate and established a technical platform for colon-targeted drug delivery, which was illuminating in this research field.
4. Conclusions Olsalazine is an effective oral treatment for both active ulcerative colitis and for maintenance of disease remission. As a prodrug, OLZ is a dimer of 5-aminosalicylic acid, which is linked by the azo bond. When taken orally, OLZ is less absorbed in stomach and small intestine, but in the colon part, the azo bond in the structure is cleaved by azoreductase produced by the microbial flora present in the colon, and 5-aminosalicylic acid is liberated. 5aminosalicylic acid is the component of OLZ for therapy of inflammatory bowel diseases, it has a selective positive effect on ulcerative colitis in inducing remission, preventing relapse and possibly reducing the risk of cancer [17]. Since the azo bond exists in the OLZ structure, OLZ itself can be used as biomaterials with colontargeted properties triggered by colon microbial flora. Based on this hypothesis, we design OLZ-linked polymer micelles containing
Author contributions Zhen-Gang Ma, Rui Ma, and Yu-Feng Zheng synthesized and characterized the polymers and micelles. Xiao-Lin Xiao, Yong-Hui Zhang, Xin-Zi Zhang, Nan Hu, Jin-Lai Gao, and De-Li Dong performed the cell culture and cytotoxic experiments. Zhi-Jie Sun designed the project and wrote the paper. Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (HEUCF201310020) from Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education, PR China. References [1] W.A. Hammond, A. Swaika, K. Mody, Pharmacologic resistance in colorectal cancer: a review, Ther. Adv. Med. Oncol. 8 (2016) 57–84. [2] S. Amidon, J.E. Brown, V.S. Dave, Colon-targeted oral drug delivery systems: design trends and approaches, AAPS PharmSciTech. 16 (2015) 731–741. [3] S. Fulda, Therapeutic exploitation of necroptosis for cancer therapy, Semin. Cell Dev. Biol. 35 (2014) 51–56. [4] L. Steinhart, K. Belz, S. Fulda, Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis, Cell Death Dis. 12 (2013) e802. [5] W. Han, L. Li, S. Qiu, Q. Lu, Q. Pan, Y. Gu, J. Luo, X. Hu, Shikonin circumvents cancer drug resistance by induction of a necroptotic death, Mol. Cancer Ther. 6 (2007) 1641–1649. [6] E. Grassilli, R. Narloch, E. Federzoni, L. Ianzano, F. Pisano, R. Giovannoni, G. Romano, L. Masiero, B.E. Leone, S. Bonin, M. Donada, G. Stanta, K. Helin, M. Lavitrano, Inhibition of GSK3B bypass drug resistance of p53-null colon carcinomas by enabling necroptosis in response to chemotherapy, Clin. Cancer Res. 19 (2013) 3820–3831. [7] C.B. Burness, E.D. Deeks, Dimethyl fumarate: a review of its use in patients with relapsing-remitting multiple sclerosis, CNS Drugs 28 (2014) 373–387. [8] R.J. Fox, M. Kita, S.L. Cohan, L.J. Henson, J. Zambrano, R.H. Scannevin, BG-12 (dimethyl fumarate): a review of mechanism of action, efficacy, and safety, Curr. Med. Res. Opin. 30 (2014) 251–262. [9] R. Loewe, T. Valero, S. KremLing, B. Pratscher, R. Kunstfeld, H. Pehamberger, P. Petzelbauer, Dimethylfumarate impairs melanoma growth and metastasis, Cancer Res. 66 (2006) 11888–11896. [10] W.G. Kirlin, J. Cai, M.J. DeLong, E.J. Patten, D.P. Jones, Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells, J. Nutr. 129 (1999) 1827–1835. [11] X. Xie, Y. Zhao, C.Y. Ma, X.M. Xu, Y.Q. Zhang, C.G. Wang, J. Jin, X. Shen, J.L. Gao, N. Li, Z.J. Sun, D.L. Dong, Dimethyl fumarate induces necroptosis in colon cancer cells through GSH depletion/ROS increase/MAPKs activation pathway, Br. J. Pharmacol. 172 (2015) 3929–3943. [12] C. Lin, H.L. Ng, W. Pan, H. Chen, G. Zhang, Z. Bian, A. Lu, Z. Yang, Exploring different strategies for efficient delivery of colorectal cancer therapy, Int. J. Mol. Sci. 16 (2015) 26936–26952.
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Please cite this article in press as: Z.-G. Ma et al., Azo polymeric micelles designed for colon-targeted dimethyl fumarate delivery for colon cancer therapy, Acta Biomater. (2016), http://dx.doi.org/10.1016/j.actbio.2016.08.021