Supporting Information. Enhanced Transdermal Drug Delivery by Transfersome-Embedded. Oligopeptide Hydrogel for Topical Chemotherapy of Melanoma

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1 Supporting Information Enhanced Transdermal Drug Delivery by Transfersome-Embedded Oligopeptide Hydrogel for Topical Chemotherapy of Melanoma Tianyue Jiang, Tong Wang, Teng Li, Yudi Ma, Shiyang Shen, Bingfang He*, Ran Mo*

2 Materials. Soybean phospholipids were purchased from Tywei Pharm. Co., Ltd. Paclitaxel (PTX) was purchased from Yew Pharmaceutical Co., Ltd. Coumarin 6 (Cou6) was provided from Sigma-Aldrich. Fmoc-Phe (Fmoc-F), Phe-Phe-Dopa (FF-Dopa) and stearyl R8H3 (R8H3-C18) were obtained from GL Biochem. The metalloprotease, WQ9-2 was expressed from Bacillus cereus and purified as described in the previous reports. 1 Cell culture. The murine melanoma B16F10 cells were cultured in Dulbecco s modified Eagle s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/ml streptomycin. The incubator condition for cell culture was maintained at 37 o C with 5% CO 2 and 90% relative humidity. All the cells used in the experiment were in the logarithmic growth phase. Tumor penetration. The three-dimensional tumor spheroids model of the B16F10 cells was formed as previously described. 2 The tumor spheroids with the diameter of about μm were incubated with Cou6-CTs and Cou6-Ts for 12 h, respectively. The tumor spheroids were then washed thrice with PBS. Finally, the fluorescent images from the surface to the middle of the spheroid were acquired by tomoscan using the confocal microscope. Cellular uptake. The B16F10 cells ( cells) were seeded in each well of a 6-well cell culture plates, and cultured at 37 C for 24 h. The cells were then incubated with Cou6-CTs and Cou6-Ts, respectively. At predetermined time points, the cells were washed thrice with PBS, and further dissociated by trypsinization. The fluorescent intensity of Cou6 in the dissociated cells was determined using a microplate reader (Tecan M1000 Pro). The quantity

3 of protein was determined using a BCA protein assay kit (Thermo). Antiproliferative effect. The B16F10 cells ( cells) were seeded in each well of a 96-well cell culture plate, and cultured for 24 h. The cells were then incubated with 200 μl of the testing samples, including Taxol, PTX-Ts, and PTX-CTs at different PTX concentrations at 37 C for 24 h, respectively. Subsequently, the cells were incubated with the methyl thiazolyl tetrazolium solution (0.5 mg/ml) at 37 C for another 4 h. After removing the solution, 150 μl of DMSO was added into each well to dissolve the cells. Finally, the absorbance (λ = 570 nm) of the DMSO solution was determined using the microplate reader. The viability of the cells after treatment was calculated according to the previous report. Apoptosis-inducing effect. The apoptosis-inducing effects of the PTX-Ts and PTX-CTs on the B16F10 cells were estimated using a caspase 3 assay kit 3 and a terminal deoxynucleotidyl transferase (TdT)-mediated dutp nick end labeling (TUNEL) kit, 4 respectively. The B16F10 cells ( cells) seeded on a sterile slide that was placed in the well of a 6-well cell culture plate, and cultured for 24 h. The cells were then incubated with the testing samples, including PTX-CTs and PTX-Ts (1 μg/ml) for 12 h. The subsequent procedures were proceeded as the manufacture s instruction described. Finally, the cells were observed by the fluorescent microscope. Animal study. The nude mice (BALB/c-nu, male, g) and the normal mice (BALB/c, male, g) were provided by the Comparative Medicine Centre of Yangzhou University. All the animal experiments were performed in accordance to the Guide for Care and Use of

4 Laboratory Animals, approved by the Animal Experimentation Ethics Committee of Nanjing Tech University. The B16F10 cells ( cells) were subcutaneously injected to the nude mice dorsum to establish the xenograft melanoma tumor mouse model. Safety evaluation. The skin of the nude mice plastered with the CTs/Gel once a day for 8 days. After 12 days, the skin was harvested for histological analysis. In addition, the activities of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP) (indicators of liver function), and blood urea nitrogen (BUN) (indicator of kidney function) in the plasma of the mice 12 days after topical treatment with the PTX-CTs/Gel (daily, 8 days) were measured using the corresponding kits, respectively. References 1. Xu, J. X.; Zhou, Z.; Wu, B.; He, B. F., Enzymatic Formation of A Novel Cell-Adhesive Hydrogel Based on Small Peptides with A Laterally Grafted l-3,4-dihydroxyphenylalanine Group. Nanoscale 2014, 6, Xue, J.; Zhao, Z.; Zhang, L.; Xue, L.; Shen, S.; Wen, Y.; Wei, Z.; Wang, L.; Kong, L.; Sun, H.; Ping, Q.; Mo, R.; Zhang, C., Neutrophil-Mediated Anticancer Drug Delivery for Suppression of Postoperative Malignant Glioma Recurrence. Nat. Nanotechnol. 2017, 12, Jiang, T.; Sun, W.; Zhu, Q.; Burns, N. A.; Khan, S. A.; Mo, R.; Gu, Z., Furin-Mediated Sequential Delivery of Anticancer Cytokine and Small-Molecule Drug Shuttled by Graphene. Adv. Mater. 2015, 27, Zhu, Q. W.; Chen, X. J.; Xu, X.; Zhang, Y.; Zhang, C.; Mo, R., Tumor-Specific

5 Self-Degradable Nanogels as Potential Carriers for Systemic Delivery of Anticancer Proteins. Adv. Funct. Mater. 2018, 28,

6 Figure S1. TEM image of the PTX-CTs. Scale bar: 50 nm.

7 Figure S2. a) Cumulative percentage of the Cou6 permeated across the skin with the hair removal from the normal mice after incubation with different Cou6 formulations over time. b) Cumulative percentage of the intradermal and transdermal Cou6 after the skin was incubated with different Cou6 formulations for 12 h. * P < 0.05, compared with Cou6-Ts (intradermal); ## P< 0.01, ### P< 0.001, compared with Cou6-Ts (transdermal)

8 Figure S3. Fluorescent images of the skin after incubation with the Cou6/Rho-CTs for 12 h. Scale bar: 200 m.

9 Figure S4. a) Fluorescent images of the B16F10 melanoma spheroids incubated with Cou6-CTs and Cou6-Ts for 12 h. The images were obtained using the confocal microscopic tomoscan imaging at the depth of 50 m from the surface of the tumor spheroid at the depth. b) Profiles of the distribution of the fluorescence signals of the Cou6 along the signified white line in the above corresponding fluorescent images.

10 Figure S5. Intracellular accumulation of the Cou6 molecules in the B16F10 cells incubated with the Cou6-Ts and Cou6-CTs for different time. ** P < 0.01, *** P <

11 Figure S6. Viability of the B16F10 cells treated with the blank CTs without PTX for 24 h.

12 Figure S7. a) Synthesis of Fmoc-FFF-Dopa from Fmoc-F and FF-Dopa by the catalysis of WQ9-2. b) TEM image of the oligopeptide hydrogel. Scale bar: 100 nm.

13 Figure S8. Cumulative percentage of the Cou6 permeated across the skin from the nude mice after incubation with Cou6-CTs/Gel over time.

14 Figure S9. a) Variation in the relative tumor volume of the B16F10 melanoma-bearing mice treated with different PTX formulations. * P < b) Variation in the body weight of the mice after treatment.

15 Figure S10. Images of the H&E-stained tissue sections of the skin with the melanoma tumor at Day 12 after treatment. Scale bars: 500 m.

16 Figure S11. Images of the H&E-stained normal tissue sections at Day 12 after treatment. Scale bar: 100 m.

17 Figure S12. Images of the H&E-stained skin sections at Day 12 after treatment with saline and CTs/Gel once a day for 8 days. Scale bar: 50 m.

18 Figure S13. Relative activities of ALT, AST, AKP (indicators for liver function), and BUN (indicators for kidney function) in the plasma of the mice at Day 12 after treatment with PTX-CTs/Gel once a day for 8 days.