Design of pH-responsive and amphiphilic pullulan-based biological macromolecule for gene delivery

Nanomedicine, particularly gene delivery, holds immense potential and offers promising therapeutic options. Non-viral systems gained attention due to their binding capacity, stability and scalability. Among these, natural polysaccharides, such as pullulan, are advantageous in terms of sustainability, biocompatibility and potential degradability. In our study, a pH-responsive and amphiphilic pullulan derivative, pullulan norbornene carboxylate (PNC-DMA) was developed, by a two-step synthetic process involving esterification with hydrophobic norbornene group and radical thiol-ene reaction to introduce a pH-dependent cationic group. The molar mass of the polymer was systematically varied to adjust its hydrophobicity and pH-responsive properties, with DS values ranging from 0.4 to 1.4. The created pullulan library exhibited apparent pK_a values between 6.5 and 7.1, which were inversely proportional to the degree of substitution (DS). This increase enhanced the polymer's affinity for genetic material, facilitated responsive complexation, provided protection against DNase, and boosted transfection efficiency. However, increased DS was associated with cytotoxicity and protein interaction. Thus, PNC-DMA(140) emerged as the most promising candidate for aqueous formulation and gene delivery in HEK293T cells, even in the presence of serum proteins. This structure-activity-relationship study paves the way for further exploration of the potential advantages of using polysaccharides in gene delivery applications.