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For nanotechnology enabled delivery of hydrophilic protein‐based drugs, several polymer‐based carrier systems have been used in the past to protect the sensitive load and to facilitate cellular uptake and crossing of biological barriers. This study uses gelatin, a natural and biodegradable macromolecule, as carrier material which is approved for several applications. Nanoprecipitation is used to form nanoparticles and to maintain the physicochemical integrity of gelatin, hydrophilic crosslinkers, e.g., paraformaldehyde, glutaraldehyde, carbodiimide, and transglutaminase are employed. However, these crosslinkers diffuse homogenously into the carrier matrix also crosslinking the polymeric matrix with the entrapped protein‐based molecules thus rendering it inactive. Hence a hydrophobic zero‐length crosslinker, diisopropylcarbodiimide, is applied to avoid diffusion into the particles. This will provide an opportunity …