Anhydride-containing oligomer-crosslinked gelatin microparticles: crosslinking process understanding using design of experiments

• Reactive anhydride-containing oligomers enable efficient crosslinking of gelatin microparticles. • Crosslinking densities spanning 34–79% were achieved through controlled process design. • Design of Experiments identified critical material attributes and process parameters governing crosslinking. • Anhydride content, base volume, and molecular anhydride distribution significantly influence network formation. • Higher crosslinking density was associated with reduced particle swelling. Crosslinked gelatin microparticles (cGM) are versatile biomaterials with applications in regenerative medicine, tissue engineering, and drug or gene delivery. In this study, we investigated the crosslinking process of pristine gelatin microparticles using new generation reactive anhydride-containing amphiphilic oligomers synthesized from stearyl acrylate, 4-acryloylmorpholine, and maleic anhydride at varying ratios. An optimal design of experiments (DoE) was employed, comprising three factors and three responses. The factors included the anhydride content applied per crosslinking reaction (AC), the volume of base, i.e. triethylamine (TEA), and the molecular anhydride distribution (MAD) of a given oligomer type, an indirect parameter reflecting the stoichiometric density of anhydride units along the oligomer chains. The responses consisted of the mean degree of crosslinking (DoC) of the particle batch, degree of swelling (DoS), and particle transparency (MT). Dynamic image analysis and light micrographs confirmed the spherical morphology of the crosslinked gelatin microparticles and demonstrated that the crosslinking step did not alter particle shape. Through DoE runs, cGM batches with a broad range of crosslinking densities (DoC: 34 – 79%) were achieved. AC exerted the strongest effect on DoC and DoC correlated with AC following a logarithmic regression. An overall correlation between DoC and DoS was evident, showing that higher crosslinking densities reduced particle swelling. The TEA volume applied during crosslinking indicated that only small amounts were still sufficient to obtain a wide range of DoC, which is a favorable outcome as minimizing TEA helps reduce potential adverse effects in the final material. Finally, through DoE study, we established that oligomer MAD as a critical material attribute for this process, as it significantly influenced all responses.