Endosomal escape is a major bottleneck for efficient non‐viral gene delivery. This paper presents the development of two novel non‐viral vectors by cross‐linking glycerol molecules with low molecular weight polyethylenimine (PEI). The vectors, namely, HG‐PEI (45 mol% glycerol content) and LG‐PEI (9 mol% glycerol content) have apparently similar DNA binding, DNA unpacking and cellular uptake abilities but differ in buffering capacity. The cellular uptake and subsequent transfection efficiency of LG‐PEI is superior to commercially available PEI 25 k. Interestingly, although the cellular uptake of HG‐PEI is higher than that of PEI 25 k, the transgene expression by HG‐PEI‐mediated transfection is very low. Inhibitor and co‐localization studies demonstrate the mechanism of endocytosis and formation of endosomes prone to lysosomal lysis of HG‐PEI polyplexes as a consequence of its weak buffering capacity. Importantly, when the lysosomal lysis is inhibited, the transgene expression of HG‐PEI‐mediated transfection increases by 9‐fold of its initial capacity which is comparable to the transfection efficiency of PEI 25 k. These results indicated that the buffering capacity of the polymers primarily impacts endosomal escape and subsequent transfection efficiency. Furthermore, this study highlights the significance of cross‐linkers in optimizing the buffering capacity when designing polymers for gene delivery.

Non‐viral gene delivery vehicles are developed by tuning the buffering capacity of PEI with glycerol molecules. Mechanistic studies reveal that the glycerol‐crosslinked PEI appears to trigger macropinocytosis for higher cellular uptake, in addition to clathrin‐ and caveolae‐mediated endocytosis. Independent of endocytic pathway, the endosomal escape of polyplexes depends on the buffering capacity of the polymers, elucidating the intracellular fate of polyplexes.