Locally Deployable Nanofiber Patch for Sequential Drug Delivery in Treatment of Primary and Advanced Orthotopic Hepatomas

Transcription

1 Supporting Information Locally Deployable Nanofiber Patch for Sequential Drug Delivery in Treatment of Primary and Advanced Orthotopic Hepatomas Jiannan Li,,, Weiguo Xu,, Di Li,, Tongjun Liu, Yu Shrike Zhang,*, Jianxun Ding,*, and Xuesi Chen*, Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun , P. R. China Department of General Surgery, The Second Hospital of Jilin University, 218 Ziqiang Street, Changchun , P. R. China Division of Engineering in Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 65 Landsdowne Street, Cambridge, Massachusetts 02139, United States Corresponding Authors * (J. Ding). * (Y. S. Zhang). * (X. Chen). Author Contributions J. Li, W. Xu, and D. Li contributed equally to this work.

2 MATERIALS AND METHODS Materials. Methoxy poly(ethylene glycol) (mpeg; number-average molecular weight (M n ) = g mol 1 ) was obtained from Sigma-Aldrich (Shanghai, P. R. China). L-lactide (L-LA) and glycolide (GA) were donated by Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, P. R. China). Diblock copolymer methoxy poly(ethylene glycol)-block-poly(lactide-co-glycolide) (mpeg-b-plga) was synthesized through the ring-opening polymerization (ROP) of L-LA and GA. Dextran (weight-average molecular weight (M W ) = 100,000 Da) was purchased from Sigma-Aldrich (Shanghai, P. R. China), and used without further purification. Triethyl benzyl ammonium chloride (TEBAC) was purchased from J&K Technology Co., Ltd. (Beijing, P. R. China). 10-Hydroxycamptothecin (HCPT) was obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, P. R. China). Tea polyphenols (TP) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, P. R. China). Chloroform (CHCl 3 ) was bought from Beijing Chemical Works (Beijing, P. R. China). Tween-80 and elastase used for membrane degradation and drug release experiments were bought from Sigma-Aldrich (Shanghai, P. R. China) and Aladdin Reagent Co., Ltd. (Shanghai, P. R. China), respectively. Alanine transaminase (ALT), aspartate aminotransferase (AST), α-fetoprotein (AFP), creatine kinase MB isoenzyme (CK-MB), and blood urea nitrogen (BUN) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Longton Co., Ltd. (Shanghai, P. R. China). The primary antibodies of proliferating cell nuclear antigen (PCNA) and caspase-3 were purchased from Abcam Reagent Co., Ltd. (Cambridge, MA, USA). The primary antibody of reactive oxygen species modulator-1 (ROMO1) was bought from Beijing Bioss Technology Co., Ltd. (Beijing, P. R. China).

3 Characterizations of mpeg and mpeg-b-plga. The commercial mpeg was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS experiment was performed on an Autoflex speed TOF/TOF mass spectrometer (Bruker Daltonics, Germany). mpeg-b-plga was characterized using proton nuclear magnetic resonance ( 1 H NMR) spectrum, Fourier-transform infrared (FT-IR) spectrum, and gel permeation chromatography (GPC). 1 H NMR spectrum of mpeg-b-plga was recorded on a 400 MHz Bruker spectrometer (Bruker; Karlsruhe, Germany) at room temperature using deuterated chloroform (CDCl 3 ) as a solvent. FT-IR was also performed on a Bio-Rad Win-IR instrument (Bio-Rad Laboratories Inc., Cambridge, MA, USA). GPC analyses were conducted on a Waters GPC system with a Waters Styragel HT6E column and a Waters 2414 refractive index detector. CHCl 3 was used as the eluent with a flow rate of 1.0 ml min 1 at 25 C. Preparation of Emulsion-Electrospun Nanofibers Loading HCPT and TP. Generally, mpeg-b-plga was dissolved in CHCl 3 at a concentration of 6.0 wt % and used as the oil phase. Dextran was dissolved in water at a concentration of 45.0 mg ml 1 as the aqueous phase. For the HCPT-loaded membrane, HCPT was dissolved in the oil phase at a concentration of 6.0 wt %. For the TP-loaded membrane, TP was dissolved in aqueous phase at a concentration of 6.0 wt %. For the dual drug-coloaded membrane, 6.0 wt % HCPT and 6.0 wt % DS were dissolved in the oil and aqueous phases at the same time, respectively. To reduce the surface tension of the oil phase, 5.0 wt % TEBAC with respect to mpeg-b-plga was used as the emulsifier and added into the PEG PLGA/CHCl 3 solution before emulsification. 1.0 ml of 45.0 mg ml 1 dextran aqueous solution was slowly dropped

4 into 11.0 ml of above oil solution, and was emulsified at a routing rate of 6,500 r min 1 for about 5 min. The above emulsified solution was loaded in a 10-mL syringe and the electrospinning parameters were set as follows: electric strength: 26 kv; inner diameter of spinneret: 0.4 mm; injection rate of solution: 1 ml h 1 ; the needle tilt angle from horizontal: 10 ; air gap distance: 15 cm. The electrospun fibers were collected on a grounded aluminum sheet. Characterizations of Membranes. The emulsion-electrospun membranes of mpeg-b-plga and dextran with or without different drugs (i.e., TP+HCPT, HCPT, or TP) were referred to as EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM, respectively, for simplicity. In order to ensure the core sheath structure of the fibers, transmission electron microscopy (TEM; JEOL; Tokyo, Japan) and confocal laser scanning microscopy (CLSM; Carl Zeiss, LSM 780; Jena, Germany) were applied in this study. For TEM analyses, pure fiber was directly deposited on a carbon-coated copper mesh. The average diameters of nanofiber and the inner core in TEM microimage were analyzed by Adobe Photoshop CS6 (Adobe, California, USA). For CLSM analysis, dextran was labeled with rhodamin-b (RhB), and then the emulsion electrospun nanofiber with RhB-dextran in the core was imaged. The surface morphologies of EEPM/TP+HCPT, EEPM/HCPT, EEPM/TP, and EEPM were analyzed by scanning electron microscopy (SEM; Inspect-F, FEI; Finland). SEM was operated in high vacuum with the accelerating voltage of 20 kv. The samples were mounted on silicon sheets and vacuum-coated with gold-palladium for s. One hundred fibers and pores of membranes were randomly selected in each group and the average diameters of fibers and pore sizes were analyzed by Adobe Photoshop CS6.

5 The hydrophilic/hydrophobic properties of membranes were analyzed by water contact angle tests. Deionized water was dropped on cm 2 membranes, which were grounded on glass slides. The contact angles were tested and recorded. For the mechanical property analyses, membranes were cut into small pieces ( cm 2 in size, approximately 0.1 mm in thickness). Tensile mechanical properties were performed using a universal test machine (Instron 4502; Instron Corporation, Springfield, NJ) at room temperature and 50% humidity. Then, the tensile strength, Young's modulus, and the elongation at break were obtained from the stress strain curves. In Vitro and In Vivo Degradation. The biodegradation of the membranes was examined by in vitro and in vivo degradation studies. Three samples of EEPM (each membrane sample was approximately 3.0 mg in mass and cm 2 in size) were immersed in 2.0 ml of phosphate-buffered saline (PBS) containing 0.05% Tween-80 without or with 0.2 mg ml 1 elastase at 37 C. Elastase is a common pancreatic enzyme that digests fibrous glycoprotein in vivo. 1,2 The incubation medium was replaced daily to maintain the enzyme activity. The samples were collected at each preset interval and were carefully rinsed, freeze-dried, and weighed. The weight loss of each membrane was carefully calculated by Eq. 1. ( W W t ) W 0 Weight Loss = 100% 0 (1) In Eq. 1, W 0 represents the initial weight of the membrane, and W t indicates the weight of the membrane during degradation at each interval. For in vivo degradation study, 3.0 mg of cm 2 -sized EEPMs were implanted subcutaneously in BALB/c mice (female, 8 12 weeks old). In the first, third, fifth, and seventh week post-implantation, the mice were euthanized, and three EEPM samples for each

6 time point were collected. The collected samples were photographed, rinsed, freeze-dried, and weighed. The weight loss of the membrane at each time point was also analyzed using Eq. 1. Furthermore, the morphologies of the degraded samples in vivo were also examined by SEM. Drug Release Study. For analyzing the drug loading content (DLC) and drug loading efficiency (DLE) of HCPT and TP, cm 2 EEPM/TP+HCPT was dissolved in CHCl 3 and the concentrations of HCPT and TP were measured by ultraviolet/visible (UV/Vis) absorbances at 370 and 276 nm, respectively. DLC and DLE were calculated by Eqs. 2 and 3, respectively. Amount of Drug in Membrane DLC (%) = 100% Amount of Drug-Loaded Membrane (2) Amount of Drug in Membrane DLE (%) = 100% Total Amount of Feeding Drug (3) For drug release study, EEPM/TP+HCPT, EEPM/HCPT, or EEPM/TP was immersed in 2.0 ml of PBS containing 0.05% Tween-80 without or with 0.2 mg ml 1 elastase. Tween-80 was used to improve the solubility of HCPT and TP in PBS or elastase solution. Three samples for each type of membrane were recorded. At each preset interval, the incubation medium was collected and replaced. The concentrations of HCPT and TP were also measured by UV/Vis. The released HCPT and TP were determined through the standard curves generated by serial concentrations of HCPT and TP. In order to compare the release rate of HCPT and TP from EEPM/TP+HCPT in elastase solution, Eq. 4 was introduced. M t n kt (4) M In Eq. 4, M t and M represent the cumulative drug release at preset interval and infinite

7 time, respectively, k is the release rate coefficient of HCPT or TP, and n is a constant related to the properties of drug. Immunofluorescence Analyses. The paraffin sections of the livers were melted and dewaxed first. The sections were immersed in boiling citrate solution and then covered by primary antibody solutions (1:50) of PCNA, ROMO1, and caspase-3. The fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Boster Biological Engineering Co., Ltd., Wuhan, P. R. China) was used to incubate the sections. Finally, the nuclei were counterstained by 4,6-diamino-2-phenylindole (DAPI; Sigma-Aldrich, Shanghai, P. R. China). The immunofluorescence was evaluated by CLSM, and the semi-quantitative analyses were performed by ImageJ (NIH, Bethesda, Maryland, USA). The fluorescent intensity of control group was determined as "1", and the ratios of fluorescent intensities of other groups and control group were calculated to obtain the relative intensities. The relative intensities of different groups were compared to evaluate the synergistic effect of HCPT and TP in the treatment. RESULTS Characterizations of mpeg and mpeg-b-plga. The MALDI-TOF MS result indicated that the M n of mpeg was g mol 1 (Figure S1A, Supporting Information). The block copolymer mpeg-b-plga was successfully synthesized in-house, and was characterized by 1 H NMR, FT-IR, and GPC analyses. Typical signals at and ppm in the 1 H NMR spectrum could be assigned to the protons in the backbone of GA ( C(O)CH 2 O ) and L-LA ( C(O)CH(CH 3 )O ), respectively (Figure S1B, Supporting Information). The degrees of polymerization (DPs) of L-LA and GA in mpeg-b-plga were

8 estimated to be 880 and 380 by the integrated area ratios of signals at and ppm attributed to the methylene protons in L-LA and GA, respectively, and 3.6 ppm assigned to the ethylene protons ( CH 2 CH 2 O ) in mpeg. The M n of mpeg-b-plga was calculated to be 86,000 g mol 1 from 1 H NMR spectrum. The absorption peaks at , , , , , and cm 1 in FT-IR spectrum indicated the stretching vibration of C(O) O with carbonyl group, C O C without carbonyl group, C C, C=O, C H, and O H, respectively (Figure S1C, Supporting Information). GPC analyses showed that the M n of mpeg-b-plga was 50,000 g mol 1, and the polydispersity index (PDI) was 1.7 (Figure S1D, Supporting Information). The difference between the M n s detected from 1 H NMR and GPC should be attributed to the different structures between mpeg-b-plga and polystyrene, which was used to form standard curve for GPC detection. REFERENCES (1) Struyvenberg, M. R.; Martin, C. R.; Freedman, S. D. Practical Guide to Exocrine Pancreatic Insufficiency - Breaking the Myths. BMC Med. 2017, 15, 29. (2) Sokocevic, D.; Bonenfant, N. R.; Wagner, D. E.; Borg, Z. D.; Lathrop, M. J.; Lam, Y. W.; Deng, B.; DeSarno, M. J.; Ashikaga, T.; Loi, R.; Hoffman, A. M.; Weiss, D. J. The Effect of Age and Emphysematous and Fibrotic Injury on the Re-Cellularization of De-Cellularized Lungs. Biomaterials 2013, 34,

9 Figure S1. Characterizations of mpeg and mpeg-b-plga. (A) MALDI-TOF MS spectrum of mpeg. (B) 1 H NMR and (C) FT-IR spectra of mpeg-b-plga. (D) GPC chromatography of mpeg-b-plga.

10 Figure S2. Distributions of nanofiber diameters and pore sizes of different membranes.

11 Figure S3. Cumulative drug release from cm 2 -sized EEPM/TP+HCPT in elastase solution over time.

12 Figure S4. Plots of M t /M against t for HCPT and TP release from EEPM/TP+HCPT in the presence of elastase.

13 Figure S5. Histopathological evaluations of organs at high resolution. (A, B) H&E staining of the heart, spleen, lung, and kidney in (A) POH model and (B) AOH model. Scale bars = 200 µm.

14 Figure S6. The orthotopic rodent hepatoma model was successfully established with the following procedure: (A) Step 1. Anesthesia and disinfection; (B) Step 2. Surgical scrub; (C) Step 3. Laparotomy; (D) Step 4. Injection of H22 cells; (E) Step 5. Sealing of injection site; (F) Step 6. Stitch of skin and subcutaneous tissues.

15 Table S1. Cumulative drug release from cm 2 -sized EEPM/TP+HCPT in elastase solution over time. Time (day) HCPT (µg) TP (µg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.86