Polymeric nanocarriers are attractive nonviral vectors for gene delivery purposes in vivo. For such applications, numerous physiological and subcellular bottlenecks have to be overcome. In that endeavor, each structural feature of nanocarriers can be optimized with respect to its corresponding challenges. Here, we focused on the interface between a model gene delivery nanocarrier and relevant constituents of the physiological environment. We screened a library of carboxymethylated dextrans (CMD) for the electrostatic coating of positively charged nanocarriers. We evaluated the jointed influence of the CMD molecular weight and charge density upon nanocarrier coating with respect to DNase, small ions, plasma proteins, red blood cells, and target cells. A total of 4 out of 26 CMD coated nanocarriers successfully passed every screening assay, but did not yield increased reporter gene expression in target cells compared to uncoated nanocarriers. The fine-tuning of CMD for nanocarrier coating yielded a relevant shortlist of candidates that will be further tested in vivo.