Cy5 labeled sirna

Cholesterol was purchased from Chengdu Kelong Chemical Company (Chengdu, China). Soybean phospholipids (SPC) were purchased from Shanghai Taiwei Chemical Company (Shanghai, China). 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) was obtained from Shanghai Advanced Vehicle Technology (AVT) Ltd. (Shanghai, China). DSPE-PEG2000-OMe and 1, 2-dioleoyl-sn-glycero-3- phosp-hoethanolamine-N-(carboxyfluorescein) (CFPE ) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Protamine sulfate (fraction X from salmon) and calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paclitaxel (PTX) and low-molecular-weight heparin (Enoxaparin sodium, LH) were purchased from Melonepharma (Dalian, China). BCL-2 siRNA, scrambled siRNA and Cy5-labeled siRNA were purchased from Gene Pharma Ltd. (Shanghai, China). The BCL-2 siRNA (siBCL-2) sense sequence is 5'-GUG AUG AAG UAC AUC CAU UdTdT-3', and the anti-sense sequence is 5'-AAU GGA UGU ACU UCA UCA CdTdT-3'. The scramble siRNA (siCTL) sense sequence is 5'-UUC UCC GAA CGU GUC ACG UTT-3', and the anti-sense sequence is 5'-ACG UGA CAC GUU CGG AGA ATT-3'.

To investigate the biodistribution of H/G(PC_8kF₆) PNPs, KB tumor–bearing mice (tumor volume of 200 to 300 mm³) were anesthetized, and the formulations with Cy5-labeled siRNA (1000 pmol/kg, total volume of 100 μl) (GenePharma, China) were administrated via tail vein injection. To monitor the distribution of PNPs, in vivo imaging was conducted on IVIS spectrum (Caliper Life Sciences, US) at 2, 8, 24, and 36 hours after injection (n = 2). Afterward, the mice were euthanized, and the tumors and major organs (heart, lung, liver, and kidney) were collected for ex vivo imaging. The radiant efficiency of the tissues was calculated using Image 3.1 software.

Imidazole-4,5-dicarbaldehyde and PEG45-PCL20-maltotriose-COO⁻ was synthesized as our previous study [17] , [18] . Polyamidoamine (PAMAM) G6 was purchased from CYD Company (Weihai, China). Spermine, PEI 25k and dimethylformamide (DMF) were obtained from Sigma-Aldrich (MA, USA). Anti-VEGFA sense strand (5′-GGAGUACCCUGAUGAGAUCdTdT-3′), antisense strand (5′-GAUCUCAUCAGGGUACUCCdTdT-3′), Cy3 and Cy5-labeled siRNA were all purchased from GenePharma (Shanghai, China). The VEGFA primer (forward 5′-AGGAGGGCAGAATCATCACG-3′ and reverse 5′-GATCCGCATAATCTGCATGGT-3′) and the GAPDH primer (forward 5′-CCAAGGTCATCCATGACAAC-3′ and reverse 5′-TCCACAGTCTTCTGAGTGGC-3′) were synthesized by and obtained from Sangon Biotech (Shanghai, China). U87MG cell line was acquired from Guo Shengrong laboratory at Shanghai Jiao Tong University (Shanghai, China). 5-week-old male Bab/c nude mice were acquired from Experimental animal center of Shanghai Jiao Tong University (Shanghai, China). The usage of animal was performed according to the guideline of Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University.