Sequential Drug Release to Modulate Collagen Synthesis and

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Controlled Release and Delivery Systems

Sequential Drug Release to Modulate Collagen Synthesis and Promote Micelle Penetration in Tumors Zhoujiang Chen, Weiping Liu, Xin Wang, Yuan Liu, and Xiaohong Li ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01600 • Publication Date (Web): 29 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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Figure 1. Characterization of drug-loaded fiber fragments. (a) SEM morphologies of FMC@Los, FMC@Tel, FMT@Los and FMT@Tel fiber fragments (insets show TEM images). (b) Percent release of MC and MT micelles after incubation of fiber fragments at 37 C in pH 7.4 and pH 6.8 buffers (n = 3). (c) TEM images of released MC from FMC@Los, MT from FMT@Los and fresh MC and MT prepared by ultrasonication. (d) DLS sizes of MC released in the presence of 2 μM and 10 and 40 mM GSH. (e) Percent release of CPT from fiber fragments after incubation at 37 C in pH 7.4 and 6.8 buffers (n = 3). 149x108mm (300 x 300 DPI)

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Figure 2. Collagen synthesis inhibition by drug-loaded fiber fragments. (a) Percent release of Los and (b) Tel from FMC@Los and FMC@Tel after incubation at 37 C in pH 7.4 and pH 6.8 buffers (n = 3). (c) Collagen synthesis by CAFs after treatment with FMC@Los and FMC@Tel in pH 6.8 buffers, using free drug (Lo and Tel), FMC and PBS treatment as control (n = 5). 80x184mm (600 x 600 DPI)


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Figure 3. Cellular uptake and toxicity of drug-loaded fiber fragments. (a) Cellular uptake efficiency of MC or MT micelles by 4T1 cells after treatment at pH 7.4 and pH 6.8 for 6 h with FMC, FMC@Los, FMC@Tel, FMT@Los, and FMT@Tel (n = 5). (b) CLSM images of 4T1 cells after treatment in pH 7.4 and pH 6.8 media with FMT@Los and FMT@Tel. The merged images with propidium iodide-counterstained images are shown in the low row. (c) Cytotoxicities to 4T1 cells after incubation in pH 7.4 and pH 6.8 media for 48 h with FMC@Los and FMC@Tel, using free Los and Tel as control (n = 5). (d) Flow cytometry analysis of 4T1 cells after treatment with FMC@Los and FMC@Tel in pH 7.4 and pH 6.8 media, compared with free Los and Tel as control. Lower left of each image, living cells; Lower right, early apoptotic cells; Upper right, late apoptotic cells; Upper left, necrotic cells. Inserted numbers indicate the percentage of cells present in each area. 149x99mm (300 x 300 DPI)



Figure 4. Collagen levels in tumor tissues. (a) The relative collagen I levels in tumors determined by Western blotting after treatment for 7 and 14 days with FMT@Los, FMT@Tel, and their combinations (FMT@L-T) of different ratios (2/1, 1/1, 1/2, and 1/3), using FMT and FMT/Los as control. (b) Masson’s trichrome and (c) immunofluorescence staining images of collagen I expressions in tumor tissues after treatment with different fiber fragments. 149x175mm (300 x 300 DPI)


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Figure 5. CLSM images to show the distribution of MT micelles in the central and distal sections of tumors after treatment for (a) 7 and (b) 14 days with FMT@Los, FMT@Tel, and FMT@L-T, compared with those of FMT and FMT/Los. 149x116mm (300 x 300 DPI)



Figure 6. Antitumor efficacy of drug-loaded fiber fragments. (a) Tumor growth, (b) body weight changes, (c) overall survival of tumor-bearing mice, (d) H&E staining images (“N” represents necrotic area, “T” represents tumor mass), (e) IHC staining images of Ki-67 and (f) HIF-1α of tumors retrieved on day 21 after intratumoral injection of FMC@Los, FMC@Tel, and FMC@L-T, compared with those of FMC, FMC/Los and PBS treatment. (g) The number of ki-67 and HIF-1α-positive cells compared with the total number of cells in IHC staining images of tumors retrieved (n = 5; *: p < 0.05 compared with other groups). 149x147mm (300 x 300 DPI)


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Figure 7. Antimetastasis effect of drug-loaded fiber fragments. (a) Visual images of Bouin-fixed lungs, (b) nodule numbers (n = 5, *: p < 0.05) and (c) H&E staining images of lung sections (“T” represents metastatic tumors) retrieved from 4T1 tumor-bearing mice after treatment for 21 days with FMC@Los, FMC@Tel, and FMC@L-T, compared with those of FMC, FMC/Los, and PBS treatment. 119x99mm (300 x 300 DPI)



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Sequential Drug Release to Modulate Collagen Synthesis and

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