Peg8000

Adeno-X 293 cells (purchased from Clontech) were plated at a density of 1 × 10⁶ cells per 60 mm culture plate one day before transfection. After digestion by PacI, 10 μg of linearized OV DNA was transfected into 60 mm culture plate with Lipofectamine 3000 transfection reagent. To collect the VPs, cells were collected one week after transfection, centrifuged at 1500 × g for 5 min at room temperature and re-suspended in 500 μL of 1 × PBS. Then cells were lysed with three consecutive ‘freeze-thaw’ cycles. To amplify virus production, the lysate was transferred to a fresh plate with 6 × 10⁶ Adeno-X 293 cells. The virus production can be further amplified by repeating the above procedure to collect more cell lysate. The supernatant containing viruses was concentrated by PEG8000 (Sangon Biotech) to a final volume of 5 mL, and then centrifuged with CsCl gradients (1.4 and 1.2 g mL⁻¹) at 100,000 × g for 8 h. To measure the virus titer, 5 × 10⁵ Adeno-X 293 cells were seeded in 12-well plate and infected with the 1 μL of purified virus. Cells were collected 6 h after infection, and the total DNA was extracted by using DNeasy Blood & Tissue kit (Qiagen). Quantitative PCR (qPCR) were performed to estimate the virus titer by using qL2 primers shown in Supplementary Table 1.

The phage morphology was visualized under a transmission electron microscope (TEM, Tecnai 12, Tecnai, Eindhoven, the Netherlands). High phage titers were concentrated using cesium chloride (CsCl, Sangon Biotech Co., Ltd, Shanghai, China) density gradient centrifugation and 10% polyethylene glycol (PEG) 8000 (Sangon Biotech Co., Ltd, Shanghai, China) precipitation, as previously described (Byun et al., 2022 (link)). Afterward, 30 μL of concentrated phage suspension was applied to a 400-mesh copper grid with a membrane and allowed to adsorb for 10 min; a filter paper was used to absorb excess liquid around the copper mesh. Then, the resulting product was dyed with 2% phosphotungstic acid (PTA, Aladdin Biochemical Technology Co., Ltd, Shanghai, China) for 10 min, and excess PTA was blotted off the filter paper and dried out under an infrared lantern.

Recombinant human insulin was purchased from Novo Nordisk (Bagsvaerd, Denmark). 1,6-Hexanediol was from Sangon Biotech (Cat No. A100159), and PEG8000 from Sangon Biotech (Cat No. A601513). Live-cell nucleus dye Hoechst from Beyotime Biotech (Cat No. C1022). Ni-NTA resin (Cat No. SA004010) was bought from Smart-Lifesciences (Changzhou, China). Palmitate was from Sigma-Aldrich (Cat No. P9417). All other chemicals were purchased from Sigma-Aldrich (Shanghai, China) or Sangon Biotech (Shanghai, China).