Pyrene

ε-caprolactone (ε-CL, Mackin, Shanghai, China) was stirred with CaH₂ for 48 h, followed by distillation under reduced pressure. 2-(Diethylamino) ethyl methacrylate (DEAEMA, Energy Chemical, Shanghai, China) and poly(ethylene glycol) methyl ether methacrylate (PEGMA, M_n = 500 Da, Energy Chemical, Shanghai, China) were passed through a column of basic alumina to remove the inhibitors and stored at −18 °C. Triethylamine (TEA, Aldrich, Saint Louis, MO, USA) and tetrahydrofuran (THF, Energy Chemical, Shanghai, China) were stirred overnight with CaH₂ and then distilled at atmospheric pressure. Toluene (Energy Chemical, Shanghai, China) was soaked with the activated 4Å molecular sieves. N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) was purchased from Energy Chemical (Shanghai, China) and used as received. Stannous octoate (Sn(Oct)₂), 2-Bromoisobutyryl bromide (BIBB), Cupric bromide (CuBr₂) and pyrene were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China) and used as received. Doxorubicin hydrochloride (DOX·HCl) which was purchased from Beijing HuaFeng United Technology Co., Ltd. (Beijing, China), was dispersed in the phosphate buffer (pH 7.4), static for 24 h, and collected by centrifuging before being used. All other reagents were used as received without further purification.

Low molecular weight chitosan (LC) with a 5 kDa molecular weight was bought from Golden-Shell Pharmaceutical Co., Ltd. (Taizhou, China) and used as received. N, N’-carbonyldiimidazole (CDI), Cys·2HCl (97%), OA (99%), DA (98%), CA (99%), Dox·HCl (98%), triethylamine (TEA, 98%) and pyrene (97%) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Hoechst 33342, paraformaldehyde (PA, 4%), MTT and GSH were procured from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were provided by Gibco BRL (Grand Island, NY, USA). Normal mouse fibroblasts cells (L929) and human lung cancer cells (A549) were provided by the Cell Bank of the Chinese Academy of Science (Shanghai, China).

CMC generally reflects the stability of micelles after solution dilution. HS15 (35 mg) and DSPE-PEG2000-L-Val (15 mg) were dissolved in 25 mL of water and diluted to yield concentrations in the range of 0.06–2000 µg/mL. Each polymer solution was transferred to a vial containing pyrene (Macklin, Shanghai, China). The emission spectra were determined by a fluorescence spectrophotometer (F-4500, Hitachi, Tokyo, Japan) at 335 nm excitation wavelength. The scanning range was 350–500 nm, the scanning speed was 240 nm/min, the excitation and emission slits were 5 nm and 2.5 nm, respectively, the voltage of the photomultiplier tube was 700 V, and the response time was 5 s. The intensities of I_373nm and I_384nm vibronic bands were evaluated, and the ratios of these intensities were plotted against the logarithm of the concentration of each sample. The CMC was taken as the intersection of two regression lines calculated from the linear portions of the graphs [27 (link)].

Twelve Bacillus strains preserved in the laboratory, including 3 strains of Bacillus subtilis, 2 strains of Bacillus atrophaeus, 2 strains of Bacillus sonorensis, 3 strains of Bacillus amyloliquefaciens, Bacillus aryabhattai, and Bacillus methylotrophicus, were used for gene screening. The pMD-19 T vector (Takara, Dalian, China) was used for cloning and sequencing. E. coli DH5α competent cells (Tsingke, Beijing, China), E. coli BL21(DE3) cells (Vazyme, Nanjing, China), the pET28a(+) vector (Yuanye, Shanghai, China), and the pET-duet vector were used for protein overexpression. Restriction enzymes Nde I, Not I, Mlu I, Sac I, Sal I, BamH I, and EcoR I (Takara) and T4 DNA ligase (Vazyme) were used for plasmid manipulation and the ligation reaction, respectively. Substrates and standard chemicals were purchased from commercial sources, including 2,2’-azinobis(3-ehtylbenzothiazolin-6-sulfnic acid) ammonium salt (ABTS) (Aladdin, China); naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, and benzo[a]pyrene (Macklin, China); and ampicillin, kanamycin, and isopropyl-beta-D-thiogalactopyranoside (IPTG) (Solarbio, Beijing, China).