A unique method for creating drug-related nanofibers (NFs) involves electrospinning. According to reported procedures, therapies utilizing hazardous solvents or synthetic chemicals have not been authorized by the most recent drug regulations. This chapter has concentrated on studies that meet the criteria for non-toxic substances. In order to provide ideal circumstances for generating NF, electrospinning, its benefits, solutions, and process conditions, followed by characterization, will focus on safety and a regulatory emphasis. The current state of characterization of NFs is being reviewed, especially regarding chemicals entrapped within them. This makes the electrospinning application of drug-loaded NFs composition-dependent; so far, much research has been carried out using toxic solvents and FDA-unapproved crosslinking systems. The parameters of processing provide a way for the technology to be potentially accepted in the pharmaceutical industry. It has been concluded that suggestions concerning reproducibility, repeatability, and erroneous conclusions arise from the poor understanding of NF characterization techniques. Static NF release models were created that can provide safety evidence. However, some dynamic models for release can give better limitations regarding nontoxic nanostructured matrices. Many countries have developed, or at least are in the process of developing, standards. Finally, issues related to new biopolymers, their applications, and trends that can facilitate the commercialization of drug-related NFs were addressed. <!----> <!----> <!----> <!---->

6-Mercaptopurine (6-MP) is a potent chemotherapeutic and immunosuppressive agent; however, its oral administration is limited by poor bioavailability and dose-dependent hepatotoxicity. This study aimed to develop and optimize a colon-targeted, pH-responsive delivery system for 6-MP using Eudragit S100-coated thiolated chitosan/casein microbeads (Eu-S100@TCh/CS) to enhance site-specific release and minimize systemic exposure. The microbeads were fabricated via emulsion cross-linking followed by solvent evaporation and optimized using a central composite design. Physicochemical characterization (FTIR spectroscopy, DSC/TGA, PXRD, and SEM) confirmed efficient drug incorporation in a molecularly dispersed state, thermal stability, and a uniform spherical morphology (541-672 µm). Swelling and dissolution studies demonstrated a clear pH-dependent behavior, with minimal swelling and negligible drug release at pH 1.2 and 6.8, and enhanced release (84.5% at 24 h) at colonic pH 7.4, corresponding to the dissolution of the Eudragit S100 coating and hydration of the polymeric core. Drug-release kinetics indicated Higuchi diffusion for uncoated beads (R ² = 0.9852; n = 0.624) and anomalous (non-Fickian) transport for the coated beads (R ² = 0.9994; n = 0.741), governed by combined diffusion and polymer relaxation mechanisms. Acute oral toxicity studies in rats revealed no significant alterations in the biochemical, hematological, or histopathological parameters, confirming systemic safety. In vitro cytotoxicity studies (MTT assay) showed enhanced antiproliferative activity of the 6-MP-loaded microbeads against HCT-116 cells compared to the free drug, attributed to improved solubility and sustained drug availability following release rather than whole-particle internalization. Blank microbeads exhibited high cell viability, confirming carrier biocompatibility. Overall, the Eu-S100@TCh/CS microbead system represents a rationally designed colon-targeted delivery platform with potential to improve local therapeutic efficacy and reduce systemic toxicity. Further studies are warranted to evaluate selectivity using normal colonic cell lines.