Supporting Information. Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer- Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and

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1 Supporting Information Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer- Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and Anticancer Efficacy Ning Li,, Hao Cai, Lei Jiang,, Jiani Hu, Ashika Bains, Jesse Hu, Qiyong Gong, Kui Luo *, and Zhongwei Gu *,,#, Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital, Sichuan University, Chengdu , China State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, Center of Advanced Pharmaceuticals and Biomaterials, China Pharmaceutical University, Nanjing , China # College of Materials Science and Engineering, Nanjing Tech University, Nanjing , China National Engineering Research Center for Biomaterials, Sichuan University, Chengdu , China Department of Radiology, Wayne State University, Detroit, Michigan 48201, United States Corresponding author : Fax: ; Phone: / luokui@scu.edu.cn; zwgu1006@hotmail.com S-1

2 Figure S1. Structure of azide-cy5.5. S1. Synthesis of Janus PEGyalted Dendrimer G2L-G3L Modified with Alkyne The Janus PEGylated dendrimer G2L-G3L-(NH2)8 were synthesized as previous described. 1-3 Under a nitrogen atmosphere, the Janus PEGylated dendrimer G2L-G3L-(NH2)8 (920 mg, 0.1 mmol), HBTU (1.52 g, 4 mmol) and HOBt (544 mg, 4 mmol) were dissolved into anhydrous DMF (25 ml). Then, 5-hexynoic acid (0.22 ml, 4 mmol) and DIPEA (2.6 ml, 16 mmol) were added under ice bath. The solution was stirred at room temperature for 72 h. After reaction, the solvents were removed. EtOAc (300 ml) was added and the organic solution was washed with NaHCO3 aq. (satd.), HCl (1 M) and NaCl aq. The solution was dried (MgSO4) and concentrated via rotary evaporation. Final product was obtained by recrystallization from EtOAc and ether. The yield of product (Janus dendrimer PEG-G2L-G3L-alkyne) was 85.2% (910 mg, brown solid). S-2

3 Scheme S1. Structure and synthesis route of Janus PEGyalted dendrimer G2L-G3L modified with alkyne. S2. Synthesis of N3-GFLG-PTX N3-GFLG-OH was synthesized as previously described. 2 Under a nitrogen atmosphere, N3- GFLG-OH (0.53 g, 1 mmol) and 4-dimethylaminopyridine (DMAP, 61 mg, 0.5 mmol) were dissolved in anhydrous DCM (100 ml). After stirring for half hour in ice bath, PTX (1.3 g, 1.5 mmol) dispersed in DCM was added. Then, the dicyclohexylcarbodiimide (DCC) dissolved in DCM was added by dropwise. The solution was stirred in ice bath for 24 h. After reaction, the solvents and insoluble substance were removed. EtOAc (300 ml) was added and the organic solution was washed with NaHCO3 aq. (satd.), HCl (1 M) and NaCl aq. The solution was dried (MgSO4) and concentrated via rotary evaporation. Final product was obtained by recrystallization with EtOAc and ether. The yield of product was 87.4% (1.2 g, white solid). ESI- MS: m/z [M + Na] +. S-3

4 Scheme S2. Structure and synthesis route of N3-GFLG-PTX. Figure S2. Mass spectrum (MALDI-TOF) of Janus dendrimer PEG-G2L-G3L-alkyne. S-4

5 A1) A2) A3) A4) B1) B2) B3) Figure S3. Changes in the size and morphology of PEGylated dendrimer-gflf-ptx prodrugbased nanoparticle incubated without enzyme over time, as measured through DLS. The realtime size of particles incubated in buffer (ph 7.4 or 5.4) for 24 h were recorded. (A1 A4) The particles incubated in buffer (ph 7.4) for 0, 4, 6, and 24 h, respectively. (B1-B3) The particles incubated in buffer (ph 5.4) for 0, 6, and 24 h, respectively. Size distributions were recorded according to intensity. S-5

6 A) T1 Cell line Cell viability (%) B) Concentration (μg/ml) 3T3 Cell line Cell viability (%) Concentration (μg/ml) Figure S4. In vitro cytotoxicity. The cytotoxicity of drug-free PEGylated dendrimer-gflg- OMe against the 4T1 (A) and 3T3 (B) cell lines. Values represent the mean ± SD (n = 5). S-6

7 Body weight shift (%) A) B) C) Figure S5. The image of all the tumors. From top to bottom, A)-C) lines present the groups of saline (control), PTX injection and PEGylated dendrimer-gflf-ptx prodrug-based nanoparticles (NPs), respectively Control PTX injection NPs Time (days) Figure S6. Shifts in mouse body weight after injection of PEGylated dendrimer-gflf-ptx prodrug-based nanoparticles (NPs), PTX injection, and saline (control) during a period of 19 days (n = 7). S-7

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