The development and application of novel nanospheres based on cationic and anionic random amphiphilic polypeptides with prolonged stability were proposed. The random copolymers, e.g., poly(L-lysine-co-D-phenylalanine) (P(Lys-co-DPhe)) and poly(L-glutamic acid-co-D-phenylalanine) (P(Glu-co-DPhe)), with different amount of hydrophilic and hydrophobic monomers were synthesized. The polypeptides obtained were able to self-assemble into nanospheres. Such characteristics as size, PDI and ζ-potential of the nanospheres were determined, as well as their dependence on pH was also studied. Additionally, the investigation of their biodegradability and cytotoxicity was performed. The prolonged stability of nanospheres was achieved via introduction of D-amino acids into the polypeptide structure. The cytotoxicity of nanospheres obtained was tested using HEK-293 cells. It was proved that no cytotoxicity up to the concentration of 500 µg/mL was observed. C-peptide delivery systems were realized in two ways: (1) peptide immobilization on the surface of P(Glu-co-DPhe) nanospheres; and (2) peptide encapsulation into P(Lys-co-DPhe) systems. The immobilization capacity and the dependence of C-peptide encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The kinetic of drug release was studied at model physiological conditions. Novel formulations of a long-acting C-peptide exhibited their effect ex vivo by increasing activity of erythrocyte Na ⁺ /K ⁺ -adenosine triphosphatase.