For transfection DNA, encoding BsAb was complexed with polyethylenimine (PEI)-Pro in Opti-Pro serum-free medium at a DNA-to-PEI ratio of 1:4 [w (μg)/v (μL)] for 15 min prior to transfecting the suspension-adapted CHO cells. For each transfection, 2 μg DNA mL−1 cells at a concentration of 3 million cells mL−1 were used. Transfected cells were maintained in chemically defined CHO medium at 37 °C, 7.5% CO2, 70% humidity with shaking at 130 rpm for 6 h, before feeding with 7.5% CHO CD EfficientFeed A, 7.5% CHO CD EfficientFeed B, and 0.4% anticlumping agent. Culture was continued at 32 °C, 7.5% CO2, 70% humidity with shaking at 130 rpm until cell viability was below 70% (7−10 days).

In the present study, a capsule system that consists of a stealth carrier based on poly(ethylene glycol) (PEG) and functionalized with bispecific antibodies (BsAbs) is introduced to examine the influence of the capsule shape and size on cellular targeting. Hollow spherical and rod-shaped PEG capsules with tunable aspect ratios (ARs) of 1, 7, and 18 were synthesized and subsequently functionalized with BsAbs that exhibit dual specificities to PEG and epidermal growth factor receptor (EGFR). Dosimetry (variation between the concentrations of capsules present and capsules that reach the cell surface) was controlled through "dynamic" incubation (i.e., continuously mixing the incubation medium). The results obtained were compared with those obtained from the "static" incubation experiments. Regardless of the incubation method and the capsule shape and size studied, BsAb-functionalized PEG capsules showed >90% specific cellular association to EGFR-positive human breast cancer cells MDA-MB-468 and negligible association with both control cell lines (EGFR negative Chinese hamster ovary cells CHO-K1 and murine macrophages RAW 264.7) after incubation for 5 h. When dosimetry was controlled and the dose concentration was normalized to the capsule surface area, the size or shape had a minimal influence on the cell association behavior of the capsules. However, different cellular internalization behaviors were observed, and the capsules with ARs 7 and 18 were, respectively, the least and most optimal shape for achieving high cell internalization under both dynamic and static conditions. Dynamic incubation showed a greater impact on the internalization of rod-shaped capsules ( approximately 58-67% change) than on the spherical capsules ( approximately 24-29% change). The BsAb-functionalized PEG capsules reported provide a versatile particle platform for the evaluation and comparison of cellular targeting performance of capsules with different sizes and shapes in vitro.