Supporting Information Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery Yongzhi Men, Shaojun Peng, Peng Yang, Qin Jiang, Yanhui Zhang, Bin Shen, Pin Dong, *, Zhiqing Pang,*, Wuli Yang,*, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, PR China State Key Laboratory of Molecular Engineering of Polymers & Department of Macromolecular Science, Fudan University, Shanghai, 200433, PR China Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 201203, PR China AUTHOR INFORMATION Corresponding Author *Telephone: +86-21-65642385. Fax: +86-21-65640293. E-mail: dongpin64@aliyun.com (P. Dong); zqpang@fudan.edu.cn (Z. Pang); wlyang@fudan.edu.cn (W. Yang). S-1
Methods S1 Procedure of IgM and IgG assay In brief, serum samples were obtained by incubation of blood samples for 30 min at room temperature followed by centrifugation at 1000 g at 4 o C for 15 min. The IgM and IgG levels in the serum were quantified using the Mouse IgM ELISA Kit and the Mouse IgG ELISA Kit purchased from Multi Science, Hangzhou, PR China. 1) The concentrated mouse IgM (IgG) standard (250 µl) + Assay Buffer(1 ) (250 µl) served as the high standard (32 ng/ml). Pipette 250 µl of assay buffer (1 ) into each tube. Use the high standard to produce a 1:1 dilution series. Mix each tube thoroughly before the next transfer. Assay Buffer (1 ) served as the zero standard (0 ng/ml). Normal serum and plasma samples required a 10,000-fold dilution. Add 300 µl of Wash Buffer (1 ) to each well, and allow the Wash Buffer in the wells for about 30 seconds before aspiration. 2) Add 100 µl of 2-fold diluted Standard in duplicate. Add 100 µl of Assay Buffer (1 ) to Blank well in duplicate. Add 90 µl of Assay Buffer (1 ) and 10 µl of diluted samples to the sample wells. Incubate at room temperature (18 to 25 o C) for 2 h on a microplate shaker set at 300 rpm. Aspirate and wash each well, repeating the process five times. 3) Wash by filling each well with 300 µl of Wash Buffer (1 ). Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. 4) Add 100 µl of diluted Detection Antibody to each well. Incubate at room temperature (18 to 25 o C) for 1 hour on a microplate shaker set at 300 rpm. Add 100µL of Substrate Solution to each well. Incubate for 5 to 30 minutes at room temperature. 5) Add 100 µl of Stop Solution to each well. Determine the optical density within 30 minutes, using a microplate reader set to 450 nm. S-2
Methods S2 In situ TUNEL staining protocol 1. Rehydration: Dewax paraffin-embedded sections were dewaxed in xylene and rehydrated in graded ethanol series to water (xyleneⅠ@ 5-10 min, xylene Ⅱ@ 5-10 min, xylene Ⅲ@ 5-10 min, absolute ethanol @ 5 min, absolute ethanol @ 5 min, 90% ethanol @ 5 min, 80% ethanol @ 5 min, 70% ethanol @ 5 min and 1X PBS @ 5 min). 2. Proteinase digestion: Permeabilization of tissue with Proteinase K (P2308 sigma) Working solution: (10-20 µg/ml in 10 mm Tris-HCl with ph 7.4-8.) Incubate for 15 min at 37 o C. Proteinase K incubation time and temperature should be adjusted for each tissue type. 3. TUNEL labeling: Preparation of TUNEL reaction mixture (for staining 10 samples and two negative controls): 1) Remove 100 µl of label solution (Vial 2) for two negative controls. 2) Add 50 µl of Enzyme solution (Vial 1) to the remaining 450 µl of label solution in Vial 2 to obtain 500 µl of TUNEL reaction mixture (50 µl per test section). 3) Mix well to equilibrate components. Rinse with PBS for 3 minutes for 2 times. Blot off excess PBS with a Kim wipe. Put 50 µl of TUNEL reaction mixture on each test section, incubate in a humidified chamber at 37 ºC in dark for 60 min. Wash sample 5 times with PBS. 4). Counterstaining: Gel Mount-DAPI: 1 ml Gel Mount (Sigma), 1 µl of 1000 DAPI stock solution (0.2 S-3
mg/ml) Drain excess solution from the slide. Apply two separate drops of Gel Mount-DAPI on the sections, then carefully lower the coverslip on sections. Let stand in dark for a few minutes, and then observe DAPI staining with UV excitation. Observe TUNEL signals with the excitation wavelength of 488 nm and the emission wavelength of 525 nm (green). S-4
Table S1. Hydrodynamic size and zeta potential of n-psbma and POEGMA nanogels measured by DLS. sample D h a (nm) PDI b ZP c n-psbma 249 0.037-9.0 ±1.2 mv POEGMA 245 0.014-5.0 ± 0.8 mv a Measured in phosphate buffer of ph 7.4 at 37 C (1 mg/ml). b Polydispersity index of the particle size. c Zeta potential of nanogels in phosphate buffer of ph 7.4 at 37 C (1 mg/ml). Table S2. Whole blood panel analysis of nanogels-treated mice at 24 h after injection (n=4). Unit Control POEGMA PSBMA WBC 10 9 cell/l 5.3 ± 1.3 6.5 ± 1.4 6.5 ± 1.3 RBC 10 12 cell/l 8.7 ± 0.9 8.7 ± 0.4 9.5 ± 1.2 HGB g/l 136 ± 10 139 ± 8 138 ± 7 HCT % 42.9 ± 2.5 42.0 ± 2.4 44.4 ± 2.5 MCV fl 49.3 ± 2.4 48.2 ± 0.4 46.1 ± 2.4 MCH pg 15.6 ± 0.4 15.9 ± 0.1 14.5 ± 0.5 MCHC g/l 317 ± 14 330 ± 10 414 ± 13 RDW % 18.5 ± 1.2 17.7 ± 0.6 19.3 ± 1.6 PLT 10 9 cell/l 696 ± 89 692 ± 96 626 ± 102 S-5
Table S3. Whole blood panel analysis of nanogels-treated mice at 48 h after injection (n=4). Unit Control POEGMA PSBMA WBC 10 9 cell/l 6.0 ± 0.2 4.4 ± 1.0 5.5 ± 0.6 RBC 10 12 cell/l 9.9 ± 0.2 10.3 ± 0.1 10.3 ± 0.1 HGB g/l 157 ± 2 160 ± 2 159 ± 2 HCT % 47.5 ±1.0 47.8 ± 0.5 48.3 ± 0.8 MCV fl 48.1 ± 0.1 46.6 ± 0.1 47.8 ± 0.4 MCH pg 15.9 ± 0.2 15.7 ± 0.1 15.8 ± 0.3 MCHC g/l 329 ± 4 326 ± 11 318 ± 8 RDW % 18.3 ± 0.3 17.5 ± 0.6 17.1 ± 0.2 PLT 10 9 cell/l 623 ± 89 559 ± 27 634 ± 55 WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW, red blood distribution width; PLT, platelets. Figure S1. Representative TEM images of n-psbma (a) and POEGMA (b) nanogels, the scale bar were 100 nm. S-6
Figure S2. In vivo NIFR imaging of POEGMA-Cy7.5 (200µg/mL, 100µL) in tumor-bearing mice (marked with dotted ellipses) at different time points after injection. Figure S3. In vivo NIFR imaging of free Cy7.5 (200 µg/ml, 100 µl) in tumor-bearing mice (marked with dotted ellipses) at 1, 2, 4 and 8 h after injection. S-7
Figure S4. (a) Fluorescence emission spectra of Cy7.5-labeled PSBMA nanogels at various concentrations. (b) Linear curve was fitted from the fluorescence intensity of Cy7.5-labeled PSBMA nanogels versus nanoparticle concentration. (λ ex = 760 nm) Figure S5. The calculated tumor inhibition rates based on tumor weight at day 15 of different groups. S-8
Figure S6. Body weight curves of each group after different treatments. (For the NS group, mice were injected with 100 µl of normal saline. For other groups, the DOX dose was 5 mg/kg and injection was performed at day 0 and day 5. S-9