Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

Figures & data

Figure 1 Schematic representation of adenovirus complexed with cationic PEG derivative (Ad5/APC) for evading anti-adenovirus neutralizing antibody (NAb).

Abbreviations: EO, ethylene oxide; AGE, allyl glycidyl ether; APC, cationic PEG derivative; Ad5, adenovirus vectors type 5.

Figure 2 Synthesis scheme of APC. (a) (Boc)2O, NaOH, MeOH:H₂O = 1:1 (v/v), RT, 12 hours. (b) NaH, EO, dioxane, 60°C, 4 days. (c) NaH, AGE, 60°C, 3 days. (d) Trifluoroacetic acid:dichloromethane = 1:4 (v/v), RT, 2 hours. (e) MeOH, RT, 2 days.

Abbreviations: APC, cationic PEG derivative; EO, ethylene oxide; AGE, allyl glycidyl ether; RT, room temperature.

Figure 3 In vitro transfection assays of Ad5 or complexes. To determine the best ratio of Ad5 and polymers, transfection assays were performed in A549 cells treated with Ad5-LacZ (1.25 × 10⁷ pfu/well) complexed with a serial dose of polymers, PEI-2k or APC (50~1000 ng). Following incubation for 24 hours after transfection, the cells was lyzed for quantity of β-galactosidae (A). Transfection assays in A549, B16, and SKOV3 treated with Ad5 alone (1.25 × 10⁷ pfu/well) or complexes (Ad5/PEI-2k or Ad5/APC) at the optimal ratio (B).

Notes: Data are represented as mean ± SD (n = 3), ***P < 0.001.

Abbreviations: Ad5, adenovirus vectors type 5; APC, cationic PEG derivative; PEI, polyethylenimine; Ad5/APC, adenovirus complexed with APC; Ad5/PEI-2k, adenovirus complexed with PEI-2k; pfu, plaque forming units; SD, standard deviation.

Table 1 Zeta potential and size distribution of naked Ad5 and complexesa

	Zeta potential (mV)	Size (nm)	PDI
Naked Ad5	−17.56 ± 0.53	115.6 ± 5.46	0.04 ± 0.037
Ad5/PEI-2k	9.64 ± 0.67	1382 ± 79.9	0.084 ± 0.11
Ad5/APC	31.23 ± 0.4	130.2 ± 0.60	0.02 ± 0.01

Notes:

a Complexes were respectively prepared at optimal ratio. Values are represent as mean ± SD, n = 3.

Abbreviations: Ad5, adenovirus vectors type 5; PDI, polydispersity index; PEI, polyethylenimine; APC, cationic PEG derivative; Ad5/APC, adenovirus complexed with APC; Ad5/PEI-2k, adenovirus complexed with PEI-2k; SD, standard deviation.

Figure 4 Transmission electron microscopy images of the naked adenovirus (A) and adenovirus complexed with APC (B).

Note: Arrow bar = 100 nm.

Abbreviation: APC, cationic PEG derivative.

Figure 5 Relative viability of (A) A549, (B) B16, and (C) SKOV3 cells treated with Ad5 (1.25 × 10⁶ pfu/well) complexed with a serial concentration of APC, PEI-25k, and PEI-2k over the range from 1 μg/mL to 20 μg/mL. Each sample has five repeated wells.

Notes: Data are represented as mean ± SD (n = 5), *P < 0.05, **P < 0.01, ***P < 0.001.

Abbreviations: Ad5, adenovirus vectors type 5; pfu, plaque forming units; PEI, polyethylenimine; APC, cationic PEG derivative; SD, standard devation.

Figure 6 Neutralizing assays in A549 cells treated with Ad5 alone (1.25 × 10⁷ pfu), Ad5/PEI-2k or Ad5/APC at the optimal ratio. Before transfection, a neutralizing effect was processed by incubation of naked Ad5 or freshly prepared complexes (Ad5/PEI-2k or Ad5/APC) and complement-inactivated blank serum or antiserum which respectively isolated from nonimmunized or immunized C57BL/6 N mice at 37°C for 1 hour. The following processes were performed as described in in vitro transfection assays.

Notes: Data are represented as mean ± SD (n = 3), ***P < 0.001.

Abbreviations: Ad5, adenovirus vectors type 5; PEI, polyethylenimine; APC, cationic PEG derivative; pfu, plaque forming units; Ad5/PEI-2k, adenovirus complexed with PEI-2k; Ad5/APC, adenovirus complexed with APC; SD, standard deviation.