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Many methods have been used to isolate and identify microplastics from biological matrices. In biological samples, Nile Red can stain undigested residues, such as fats, soaps, and gels formed during organic matter removal, hindering the identification of fluorescent microplastics (≥2 μm). Thus, adjustments on sample preparation (e.g., fat removal) are required for the accurate identification of Nile Red stained microplastics. Multiples tests allowed to identify that digestion with 10% KOH at 60 °C for 24 h, followed by treatments with boiling water, acetone, and staining, produced good results in fourteen biological samples, including vertebrates and invertebrates. Digestion efficiencies were 94-100%, except for feces, which were 87%. Recovery rates of spiked microplastics were 97-100%, and few effects were observed in the infrared spectra and carbonyl index of seven polymers, with only the occasional yellowing suggesting surface changes. Filtration rates were improved by reducing the amount of sample. Small fluorescent microplastics could be identified in all samples under the microscope. Overall, the proposed method was efficient in removing natural organic matter from biological samples for Nile Red staining, requiring minimal sample handling, improving sample throughput, and allowing quantification of fluorescent microplastics in biological samples.

The present study reports the fabrication of dissolvable microneedle (MN) patches using pullulan (PL), a water-soluble polysaccharide with excellent film-forming ability, for the transdermal administration of insulin, envisioning the non-invasive treatment of diabetes. PL MNs patches were successfully prepared by micromoulding and revealed good thermal stability (Tdmax = 294 °C) and mechanical properties (>0.15 N needle−1), penetrating skin up to 381 μm depth, as revealed by in vitro skin tests. After application into human abdominal skin in vitro, the MNs dissolved within 2 h releasing up to 87% of insulin. When stored at 4, 20 and 40 °C for 4 weeks, insulin was able to retain its secondary structure, as shown by circular dichroism spectropolarimetry. The prepared PL MNs were non-cytotoxic towards human keratinocytes, being suitable for skin application. These findings suggest that PL MNs have potential to deliver insulin transdermally, thus avoiding its subcutaneous administration.